Adsorbing pathogen-inactivating compounds with porous particles immobilized in a matrix

ABSTRACT

Methods and devices are provided for reducing the concentration of low molecular weight compounds in a biological composition containing cells while substantially maintaining a desired biological activity of the biological composition. The device comprises highly porous adsorbent particles, and the adsorbent particles are immobilized by an inert matrix. The matrix containing the particles is contained in a housing, and the particles range in diameter from about 100 μm to about 1500 μm. The device can be used to adsorb and remove a pathogen-inactivating compounds from a biological composition such as a blood product.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 11/243,822,filed Oct. 4, 2005 now U.S. Pat. No. 7,611,831, which is acontinuation-in-part of application Ser. No. 10/011,202, filed Dec. 7,2001, now abandoned, which is a continuation of application Ser. No.09/112,400, filed Jul. 8, 1998, now abandoned, which is acontinuation-in-part of application Ser. No. 09/003,113, filed Jan. 6,1998, now abandoned. This application is also a continuation-in-part ofapplication Ser. No. 10/016,323, filed Dec. 10, 2001, now U.S. Pat. No.6,951,713, which is a continuation of application Ser. No. 09/112,068,filed Jul. 8, 1998, now abandoned, which is a continuation-in-part ofapplication Ser. No. 09/003,113, filed Jan. 6, 1998, now abandoned. Allof the applications disclosed above are incorporated by reference intheir entirety.

TECHNICAL FIELD

The present invention relates to methods and devices for the reductionof compounds in biological compositions. The compounds have a molecularweight ranging from about 100 g/mol to about 30,000 g/mol.

BACKGROUND ART

An extensive body of research exists regarding the removal of substancesfrom blood products. The bulk of this research is directed at white cellreduction. See, e.g., M. N. Boomgaard et al., Transfusion 34:311 (1994);F. Bertolini et al., Vox Sang 62:82 (1992); and A. M. Joustra-Dijkhuiset al., Vox Sang 67:22 (1994). Filtration of platelets is the mostcommon method used in white cell reduction of platelet concentrates.See, e.g., M. Böck et al., Transfusion 31:333 (1991) (Sepacell PL-5A,Asahi, Tokyo, Japan); J. D. Sweeney et al., Transfusion 35:131 (1995)(Leukotrap PL, Miles Inc., Covina, Calif.); and M. van Marwijk et al.,Transfusion 30:34 (1990) (Cellselect, NPBI, Emmer-Compascuum, TheNetherlands; Immugard Ig-500, Terumo, Tokyo, Japan). These currentfiltration mechanisms, however are not amenable for the removal ofrelatively low molecular weight compounds including for examplepsoralens, psoralen photoproducts and other compounds commonly used intreating biological fluids.

The process of adsorption has been used to isolate selective bloodcomponents onto phospholipid polymers. For example, several copolymerswith various electrical charges have been evaluated for theirinteractions with blood components, including platelet adhesion andprotein adsorption. K. Ishihara et al., J. Biomed. Mat. Res. 28:1347(1994). Such polymers, however, are not designed for the adsorption oflow molecular weight compounds.

Various dialysis means are able to remove low molecular weight compoundsfrom plasma and whole blood. For example, dialysis can successfullyremove low molecular weight toxins and pharmaceutical compounds. Thus,dialysis might be used to remove, for example, psoralens and psoralenphotoproducts from blood products. Unfortunately, current dialysisprocedures involve very complicated and expensive devices. As such, theuse of dialysis machines would not be practical for the decontaminationof a large volume of blood products.

The use of polystyrene divinylbenzene, silica gel, and acrylesterpolymers for the adsorption of methylene blue has previously beendescribed. For example, PCT Publication No. WO 91/03933 describes batchstudies with free adsorbent resin (e.g., Amberlites (Rohm and Haas(Frankfurt, Germany) and Bio Beads (Bio-Rad Laboratories (Munich,Germany)). Without very careful removal of the adsorbent resins afterexposure to the blood product, however, these methods create the risk oftransfusion of the resin particles.

In addition, devices and processes for the removal of leukocytes andviral inactivation agents (e.g., psoralens, hypericin, and dyes such asmethylene blue, toluidine blue, and crystal violet) have also beendisclosed. Specifically, PCT Publication No. WO 95/18665 describes afilter comprising a laid textile web which includes a mechanicallystable polymeric substrate. The web itself comprises interlocked textilefibers forming a matrix with spaces and fibrillated particles disposedwithin the spaces. However, this device causes a significant decrease inthe Factor XI activity, which may render the treated product unsuitablefor its intended use.

Simpler, safer and more economical means for reducing the concentrationof low molecular weight compounds in a biological composition containingcells while substantially maintaining the biological activity of thetreated biological composition containing cells are therefore needed.

DISCLOSURE OF THE INVENTION

The present invention provides devices for reducing the concentration ofcompounds in biological compositions containing cells. The devicesinclude an adsorption medium comprised of particles immobilized by aninert matrix and are of a batch configuration. Typically, the compoundsreduced in biological compositions using the device have molecularweights ranging from about 100 g/mol to about 30,000 g/mol. Thebiological composition containing cells includes, for example, cellssuspended in a biological medium, such as plasma or tissue culturemedia. The biological activity of the biological composition issubstantially maintained after contact with such devices.

Exemplary compounds include pathogen inactivating compounds, dyes,thiols, plasticizers and activated complement. Devices are provided thatcomprise a three dimensional network of adsorbent particles immobilizedby an inert matrix. This immobilization reduces the risk of leakage ofloose adsorbent particles into the blood product. Furthermore,immobilization of the adsorbent particles by an inert matrix simplifiesmanufacturing by reducing problems associated with handling looseadsorbent particles. Immobilization of the adsorbent particles may alsoenhance the ability of the adsorbent particles to adsorb compounds inbiological compositions containing cells without mechanical damage tothe cells.

The present invention provides a device for reducing the concentrationof low molecular weight compounds in a biological composition containingcells such that the cells are suitable for their intended use.

In one embodiment, the device comprises an inert matrix containinghighly adsorbent particles having a diameter ranging from about 100 μmto about 1500 μm. The cells in the biological composition treated withthe device substantially maintain their desired biological activity. Thedevice is for use in a batch process.

In another embodiment, the device further comprises a container that iscompatible with the biological composition.

In another embodiment, the adsorbent particles have a surface areagreater than about 750 m²/g.

In another embodiment, the adsorbent particles have a surface areabetween about 1000 m²/g and 3000 m²/g.

In another embodiment, the adsorbent particles are polyaromaticadsorbent particles.

In another embodiment, the polyaromatic adsorbent particles possesssuperior wetting properties.

In another embodiment, the polyaromatic adsorbent particles comprise ahypercrosslinked polystyrene network.

In another embodiment, the adsorbent particles are activated carbonparticles.

In another embodiment, the activated carbon particles are derived from asynthetic source.

In another embodiment, the low molecular weight compound is a quencher.

In another embodiment, the activated carbon particles are roughlyspherical and are of a diameter ranging from about 300 μm to about 900μm.

In another embodiment, the inert matrix is a synthetic polymer fiber.

In another embodiment, the synthetic polymeric fiber comprises a polymercore with a high melting temperature surrounded by a sheath with a lowermelting temperature.

In another embodiment, the inert matrix is a particulate network.

In another embodiment, the particulate network comprises polyethyleneparticles.

In another embodiment, the adsorbent particles are immobilized in thematrix.

In another embodiment, the low molecular weight compound is a nucleicacid targeting agent or a photosensitizer.

In another embodiment, the low molecular weight compound is an acridinederivative, a psoralen derivative or a dye.

In another embodiment, the low molecular weight compound is a biologicalresponse modifier.

In another embodiment, the biological response modifier is activatedcomplement.

In another embodiment, the low molecular weight compound is a quencher.

In another embodiment, the low molecular weight compound is a polyaminederivative.

In another embodiment, the desired biological activity maintained by thecells is suitability for infusion.

In another embodiment, the biological composition comprises platelets.

In another embodiment, treatment of the biological composition with adevice over a period of about 5 days results in less than about a 10%loss in platelet count.

In another embodiment, the device comprises an inert matrix containinghighly adsorbent particles having a diameter ranging from about 100 μmto about 1500 μm. The cells in the biological composition treated withthe device substantially maintain their desired biological activity. Thedevice is for use in a batch process. The biological compositioncomprises platelets. Treatment of the biological composition with thedevice over a period of about 5 days results in less than about a 10%loss in platelet count. The device further comprises a container that iscompatible with the biological composition, and wherein the inert matrixis a synthetic polymer fiber comprising a polymer core with a highmelting point surrounded by a sheath with a lower melting temperature,and wherein the adsorbent particles are polyaromatic adsorbentscomprising a hypercrosslinked polystyrene network, and wherein thesurface area of the particles is greater than about 750 m²/g, andwherein the adsorbent particles are immobilized in the matrix. Anexample of desired biological activity maintained by the cells includes,without limitation, suitability for infusion. Examples of low molecularweight compounds reduced by the device include, without limitation, anacridine derivative, a psoralen derivative, a dye and a biologicalresponse modifier such as activated complement. A device of thisembodiment, as an example, may be used to treat the biologicalcomposition at a temperature of 22° C. for between about 0.5 hour andabout 7 days.

In another embodiment, the biological composition comprises red bloodcells.

In another embodiment, treatment of the biological composition with thedevice over a period of up to 35 days results in less than about 1%hemolysis.

In another embodiment, the device comprises an inert matrix containinghighly adsorbent particles. The cells in the biological compositiontreated with the device substantially maintain their desired biologicalactivity. The device is for use in a batch process. The biologicalcomposition comprises red blood cells. Treatment of the biologicalcomposition with the device over a period of up to 35 days results inless than about 1% hemolysis. The device further comprises a containerthat is compatible with the biological composition, and wherein theinert matrix is a synthetic polymer fiber comprising a polymer core witha high melting point surrounded by a sheath with a lower meltingtemperature, and wherein the adsorbent particles are activated carbonparticles derived from a synthetic source, and wherein the diameter ofthe particles ranges from about 300 μm to about 900 μm, and wherein thesurface area of the particles is greater than about 750 m²/g, andwherein the adsorbent particles are immobilized in the matrix. Anexample of desired biological activity maintained by the cells includes,without limitation, suitability for infusion. Examples of low molecularweight compounds reduced by the device include, without limitation, anacridine derivative, a psoralen derivative, a dye, a biological responsemodifier such as activated complement, and a quencher such asglutathione.

The present invention also provides a method for reducing theconcentration of a low molecular weight compound in a biologicalcomposition containing cells, wherein the cells in the biologicalcomposition treated with a device substantially maintain their desiredbiological activity after treatment for between 0.5 hour and 5 weeks.

In one embodiment, the device comprises an inert matrix containinghighly adsorbent particles having a diameter ranging from about 100 μmto about 1500 μm. The cells in the biological composition treated withthe device substantially maintain their desired biological activity. Thedevice is for use in a batch process.

In another embodiment, the device comprises an inert matrix containinghighly adsorbent particles having a diameter ranging from about 100 μmto about 1500 μm. The cells in the biological composition treated withthe device substantially maintain their desired biological activity. Thedevice is for use in a batch process. The biological compositioncomprises platelets. Treatment of the biological composition with thedevice over a period of about 5 days results in less than about a 10%loss in platelet count. The device further comprises a container that iscompatible with the biological composition, and wherein the inert matrixis a synthetic polymer fiber comprising a polymer core with a highmelting point surrounded by a sheath with a lower melting temperature,and wherein the adsorbent particles are polyaromatic adsorbentscomprising a hypercrosslinked polystyrene network, and wherein thesurface area of the particles is greater than about 750 m²/g, andwherein the adsorbent particles are immobilized in the matrix.

In another embodiment, the device comprises an inert matrix containinghighly adsorbent particles. The cells in the biological compositiontreated with the device substantially maintain their desired biologicalactivity. The device is for use in a batch process. The biologicalcomposition comprises red blood cells. Treatment of the biologicalcomposition with the device over a period of up to 35 days results inless than about 1% hemolysis. The device further comprises a containerthat is compatible with the biological composition, and wherein theinert matrix is a synthetic polymer fiber comprising a polymer core witha high melting point surrounded by a sheath with a lower meltingtemperature, and wherein the adsorbent particles are activated carbonparticles derived from a synthetic source, and wherein the diameter ofthe particles ranges from about 300 μm to about 900 μm, and wherein thesurface area of the particles is greater than about 750 m²/g, andwherein the adsorbent particles are immobilized in the matrix.

In another embodiment, the cells in the biological composition treatedwith the device substantially maintain their desired biological activityafter being treated with the device for between about 1.0 hour and 5weeks.

In another embodiment, the low molecular weight compound is an acridinederivative, a psoralen derivative or a dye.

In another embodiment, the low molecular weight compound is a biologicalresponse modifier.

In another embodiment, the biological response modifier is activatedcomplement.

In another embodiment, the biological composition is treated with thedevice at a temperature of about 22° C. for between about 0.5 and about36 hours.

In another embodiment, the biological composition is treated with thedevice at a temperature of about 22° C. for between about 0.5 and about24 hours.

In another embodiment, the biological composition is treated with thedevice at a temperature of about 22° C. for between about 0.5 and about12 hours.

In another embodiment, the biological composition is treated with thedevice at a temperature of about 22° C. for between about 0.5 and about24 hours and is subsequently treated with the device at a temperature ofabout 4° C. for up to 5 weeks.

The present invention also provides a composition containing cells,wherein the biological composition is suitable for infusion. Thebiological composition is produced by treating a biological compositioncontaining cells with a device for a period between about 0.5 hour and 5weeks.

In one embodiment, the device comprises an inert matrix containinghighly adsorbent particles having a diameter ranging from about 100 μmto about 1500 μm. The cells in the biological composition treated withthe device substantially maintain their desired biological activity. Thedevice is for use in a batch process.

In another embodiment, the device comprises an inert matrix containinghighly adsorbent particles having a diameter ranging from about 100 μmto about 1500 μm. The cells in the biological composition treated withthe device substantially maintain their desired biological activity. Thedevice is for use in a batch process. The biological compositioncomprises platelets. Treatment of the biological composition with thedevice over a period of about 5 days results in less than about a 10%loss in platelet count. The device further comprises a container that iscompatible with the biological composition, and wherein the inert matrixis a synthetic polymer fiber comprising a polymer core with a highmelting point surrounded by a sheath with a lower melting temperature,and wherein the adsorbent particles are polyaromatic adsorbentscomprising a hypercrosslinked polystyrene network, and wherein thesurface area of the particles is greater than about 750 m²/g, andwherein the adsorbent particles are immobilized in the matrix.

In another embodiment, the device comprises an inert matrix containinghighly adsorbent particles. The cells in the biological compositiontreated with the device substantially maintain their desired biologicalactivity. The device is for use in a batch process. The biologicalcomposition comprises red blood cells. Treatment of the biologicalcomposition with the device over a period of up to 35 days results inless than about 1% hemolysis. The device further comprises a containerthat is compatible with the biological composition, and wherein theinert matrix is a synthetic polymer fiber comprising a polymer core witha high melting point surrounded by a sheath with a lower meltingtemperature, and wherein the adsorbent particles are activated carbonparticles derived from a synthetic source, and wherein the diameter ofthe particles ranges from about 300 μm to about 900 μm, and wherein thesurface area of the particles is greater than about 750 m²/g, andwherein the adsorbent particles are immobilized in the matrix.

The adsorbent particles may be immobilized within a sintered matrixformed from polymeric particulate material.

Also disclosed is a pathogen-inactivating compound adsorption systemconfigured to remove the pathogen-inactivating compound in a flowprocess. The diameter of adsorbent particles of this system ranges fromabout 1 micron to about 200 micron, and the adsorbent particles areimmobilized within a sintered matrix formed from polymeric particulatematerial. The system further comprises a particle retention mediumdownstream of the adsorption medium, wherein the particle retentionmedium retains particles shed from the adsorption medium.

The present invention also provides a device for reducing theconcentration of small organic compounds in a blood product whilesubstantially maintaining a desired biological activity of the bloodproduct.

In one embodiment, the device comprises highly porous adsorbentparticles, wherein the adsorbent particles are immobilized by an inertmatrix.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 diagrammatically depicts a perspective view of one embodiment ofa fiber, indicating its inner core and outer sheath, that forms thefiber networks of the immobilized adsorbent media.

FIG. 2 schematically represents a portion of one embodiment of theimmobilized adsorbent media of the present invention.

FIG. 3 diagrammatically represents a cross-sectional view of oneembodiment of immobilized adsorbent media in which the adsorbent beadsare secured to fibers that make up the fiberized resin.

FIG. 4 diagrammatically represents a cross-sectional view of oneembodiment of immobilized adsorbent media in which the adsorbent beadsare immobilized within the fibers of the immobilized adsorbent media andthe heat seals that encompass samples of fiberized resin.

FIG. 5 is a graph showing a comparison of adsorption kinetics forremoval of aminopsoralens from platelets with Dowex® XUS-43493 andAmberlite® XAD-16 HP loose adsorbent beads and immobilized adsorbentmedia containing Amberlite® XAD-16.

FIG. 6 is a graph showing a comparison of adsorption kinetics forremoval of aminopsoralens from platelets with immobilized adsorbentmedia containing Amberlite® XAD-16 and immobilized adsorbent media withthe two different loadings of activated charcoal. Fiberized XAD-16 datais represented by circles, solid line; fiberized AQF-500-B as squares,short dashes; and, fiberized AQF-375-B as triangle, long dashes.

FIG. 7 is a graph showing a comparison of the adsorption kinetics forremoval of aminopsoralens from platelets with p(HEMA)-coated anduncoated Dowex® XUS-43493 beads.

FIG. 8 is a graph showing a comparison of the effect of pre-treatmentwith solutions containing glycerol on the relative adsorption capacityof Amberlite® XAD-16 and Dowex® XUS-43493 for aminopsoralens.

FIG. 9 is a graph showing a comparison of the effect of wetting solutionon 4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen adsorptioncapacities for dried adsorbent in 100% plasma for Amberlite® XAD-16(bottom) and Dowex® XUS-43493 (top); the samples that were not wet in anethanol solution are labeled “No Tx”. Adsorption capacities are reportedas percentages relative to the capacity of optimally wet adsorbent.

FIG. 10 is a graph showing a comparison of adsorption of aminopsoralensover a 3-hour period from plasma using Amberlite® XAD-16 wet in severaldifferent solutions.

FIG. 11 is a graph showing a comparison of the kinetics of adsorption ofmethylene blue over a 2-hour period from plasma.

FIG. 12 depicts the chemical structures of acridine, acridine orange,9-amino acridine, and 5-[(β-carboxyethyl)amino]acridine.

FIG. 13 is a graph showing plots the data for adenine capacity (y-axis)and 5-[(β-carboxyethyl)amino]acridine capacity (x-axis) for variousresins.

FIGS. 14A and 14B is a graph showing a comparison of the adsorptionkinetics for removal of 5-[(β-carboxyethyl)amino]acridine with Dowex®XUS-43493 and Purolite® MN-200 and Amberlite® XAD-16 HP.

FIG. 15 is a graph showing a comparison of the adsorption kinetics forremoval of 9-amino acridine and acridine orange with Dowex® XUS-43493.

FIG. 16 is an illustration of a batch configuration for the immobilizedadsorption device (IAD).

FIG. 17 is a graph showing a comparison of the adsorption isotherms forvarious Ambersorbs as compared to Purolite MN-200.

FIG. 18 is a graph showing a comparison of the levels of5-[(β-carboxyethyl)amino]acridine and GSH in the supernatant of 300 mLPRBC units with continued or terminated exposure after 24 hours to afiberized Pica G277 IAD (500 g/m²) over 4 weeks of storage at 4° C.

FIG. 19 is a graph showing the effect of enclosure material onadsorption kinetics for 5-[(β-carboxyethyl)amino]acridine in PRBCs.

FIG. 20 is a graph showing a comparison of percent hemolysis for theadsorbent devices containing non-immobilized and immobilized adsorbentparticle Purolite MN-200.

FIG. 21 is a graph showing a comparison of percent hemolysis for thenon-immobilized and immobilized adsorbent particle Pica G-277 activatedcarbon.

FIG. 22 is a graph showing kinetics for removal 4′-(4-amino-2-oxa)butyl-4,5′,8 trimethylpsoralen from platelet concentrates.

FIG. 23 is a graph showing psoralen adsorption kinetics for fiber matrixIAD (AQF, squares) and particulate matrix IAD (Porex, triangles).

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention provides devices for reducing the concentration ofcompounds in biological compositions containing cells. The devicesinclude an adsorption medium comprised of particles immobilized by aninert matrix and are of a batch configuration. Typically, the compoundsreduced in biological compositions using the device have molecularweights ranging from about 100 g/mol to about 30,000 g/mol. Thebiological composition containing cells includes, for example, cellssuspended in a biological medium, such as plasma or tissue culturemedia. The biological activity of the biological composition issubstantially maintained after contact with such devices.

Exemplary compounds include pathogen inactivating compounds, dyes,thiols, plasticizers and activated complement. Devices are provided thatcomprise a three dimensional network of adsorbent particles immobilizedby an inert matrix. This immobilization reduces the risk of leakage ofloose adsorbent particles into the blood product. Furthermore,immobilization of the adsorbent particles by an inert matrix simplifiesmanufacturing by reducing problems associated with handling looseadsorbent particles. Immobilization of the adsorbent particles may alsoenhance the ability of the adsorbent particles to adsorb compounds inbiological compositions containing cells without mechanical damage tothe cells.

Definitions

The term “acridine derivatives” refer to a chemical compound containingthe tricyclic structure of acridine (dibenzo[b,e]pyridine;10-azanthracene). The compounds have an affinity for (and can bind) tonucleic acids non-covalently through intercalation. The term“aminoacridine” refers to those acridine compounds with one or morenitrogen-containing functional groups. Examples of aminoacridinesinclude 9-amino acridine and acridine orange (depicted in FIG. 12).

The term “adsorbent particle” broadly refers to any natural or syntheticparticulate material which is capable of interacting with molecules in aliquid thus allowing the molecule to be removed from the liquid.Examples of naturally occurring adsorbents include but are not limitedto activated carbon, silica, diatomaceous earth, and cellulose. Examplesof synthetic adsorbents include but are not limited to polystyrene,polyacrylics, and carbonaceous adsorbents. Adsorbent particles are oftenporous, often possess high surface areas, and may be modified with avariety of functional groups (e.g. ionic, hydrophobic, acidic, basic)which can affect how the adsorbent interacts with molecules.

The term “aromatic,” “aromatic compounds,” and the like refer broadly tocompounds with rings of atoms having delocalized electrons. Themonocyclic compound benzene (C₆H₆) is a common aromatic compound.However, electron delocalization can occur over more than one adjacentring (e.g., naphthalene (two rings) and anthracene (three rings)).Different classes of aromatic compounds include, but are not limited to,aromatic halides (aryl halides), aromatic heterocyclic compounds,aromatic hydrocarbons (arenes), and aromatic nitro compounds (aryl nitrocompounds).

The term “biocompatible coating” refers broadly to the covering of asurface (e.g., the surface of a polystyrene bead) with a hydrophilicpolymer that when in contact with a blood product does not result in aninjurious, toxic, or immunological response and renders the surface morebiocompatible by decreasing cell adhesion, decreasing protein adsorptionor improving cell function. Suitable coatings are biocompatible if theyhave minimal, if any, effect on the biological material to be exposed tothem. By “minimal” effect it is meant that no significant biologicaldifference is seen compared to the control. In preferred embodiments,biocompatible coatings improve the surface hemocompatibility ofpolymeric structures. For example, poly(2-hydroxyethyl methacrylate)(pHEMA) is frequently used for the coating of materials used in medicaldevices (e.g., blood filters).

The term “biocompatible housing” refers broadly to containers, bags,vessels, receptacles, and the like that are suitable for containing abiological material, such as, for example, compositions containingplatelets or red blood cells. Suitable containers are biocompatible ifthey have minimal, if any, effect on the biological material to becontained therein. By “minimal” effect it is meant that no significantdifference is seen in blood product function compared to the control asdescribed herein, for red blood cells, platelets and plasma. Thus, bloodproducts may be stored in biocompatible housings prior to transfusion toa recipient. In a preferred embodiment, biocompatible housings are bloodbags, including a platelet storage container or red blood cell storagecontainer.

The term “container that is compatible with the biological composition”refers to a container that is suitable for holding a biologicalcomposition containing cells, such as, for example, cell culturecompositions, as well as compositions containing platelets or red bloodcells. Such containers have a minimal effect on a biological compositioncontaining cells. Examples of such containers include, withoutlimitation, cell culture plates, cell culture bottles and blood bags.

The term “biological fluids” include media from cell cultures, syntheticmedia for the storage of cells, human or non-human whole blood, plasma,platelets, red blood cells, leukocytes, serum, lymph, saliva, milk,urine, or products derived from or containing any of the above, alone orin mixture, with or without a chemical additive solution. Preferably,the fluid is blood or a blood product with or without a chemicaladditive solution, more preferably plasma, platelets and red bloodcells, most preferably apheresis plasma, red blood cells and platelets.

The term “blood bag” refers to a form of blood product container.

The term “blood product” refers to the fluid and/or associated cellularelements and the like (such as erythrocytes, leukocytes, platelets,etc.) that pass through the body's circulatory system; blood productsinclude, but are not limited to, blood cells, platelet mixtures, serum,and plasma. The term “platelet mixture” refers to one type of bloodproduct wherein the cellular element is primarily or only platelets. Aplatelet concentrate (PC) is one type of platelet mixture where theplatelets are associated with a smaller than normal portion of plasma.In blood products, synthetic media may make up that volume normallyoccupied by plasma; for example, a platelet concentrate may entailplatelets suspended in 35% plasma/65% synthetic media. Frequently, thesynthetic media comprises phosphate.

The term “blood separation means” refers broadly to a device, machine,or the like that is able to separate blood into blood products (e.g.,platelets and plasma). An apheresis system is one type of bloodseparation means. Apheresis systems generally comprise a bloodseparation device, an intricate network of tubing and filters,collection bags, an anticoagulant, and a computerized means ofcontrolling all of the components.

The term “crosslinked” refers broadly to linear molecules that areattached to each other to form a two- or three-dimensional network. Forexample, divinylbenzene (DVB) serves as the crosslinking agent in theformation of styrene-divinylbenzene copolymers. The term alsoencompasses “hypercrosslinking” in which hypercrosslinked networks areproduced by crosslinking linear polystyrene chains either in solution orin a swollen state with bifunctional agents. A variety of bifunctionalagents can be used for cross-linking (for example, see Davankov andTsyurupa, Reactive Polymers 13:24-42 (1990); Tsyurupa et al., ReactivePolymers 25:69-78 (1995).

The term “cyclic compounds” refers to compounds having one (i.e., amonocyclic compounds) or more than one (i.e., polycyclic compounds) ringof atoms. The term is not limited to compounds with rings containing aparticular number of atoms. While most cyclic compounds contain ringswith five or six atoms, rings with other numbers of atoms (e.g., threeor four atoms) are also contemplated by the present invention. Theidentity of the atoms in the rings is not limited, though the atoms areusually predominantly carbon atoms. Generally speaking, the rings ofpolycyclic compounds are adjacent to one another; however, the term“polycyclic” compound includes those compounds containing multiple ringsthat are not adjacent to each other.

The term “dye” refers broadly to compounds that impart color. Dyesgenerally comprise chromophore and auxochrome groups attached to one ormore cyclic compounds. The color is due to the chromophore, while thedying affinities are due to the auxochrome. Dyes have been grouped intomany categories, including the azin dyes (e.g., neutral red, safranin,and azocarmine B); the azo dyes; the azocarmine dyes; the dephenymethanedyes; the fluorescein dyes; the ketonimine dyes; the rosanilin dyes; thetriphenylmethane dyes; the phthalocyanines; and, hypericin. It iscontemplated that the methods and devices of the present invention maybe practiced in conjunction with any dye that is a cyclic compound.

The term “fiberized resin” generally refers to immobilization ofadsorbent material, including for example, resins entrapped in orattached to a fiber network. In one embodiment, the fiber network iscomprised of polymer fibers. In another embodiment, the fibers consistof a polymer core (e.g., polyethylene terephthalate [PET]) with a highmelting point surrounded by a polymer sheath (e.g., nylon or modifiedPET) with a relatively low melting temperature. Fiberized resin may beproduced by heating the fiber network, under conditions that do notadversely affect the adsorbent capacity of the resin to a significantdegree (temperature sufficient to melt the sheath but not the core).Where the resin comprises beads, heating is performed such that theadsorbent beads become attached to the outer polymer sheath to create“fiberized beads”. By producing fiberized resin containing a knownamount of adsorbent beads per defined area, samples of fiberized resinfor use in the removal of cyclic compounds (e.g., psoralens, and, inparticular, aminopsoralens) and other products can be obtained bycutting a defined area of the fiberized resin, rather than weighing theadsorbent beads.

The term “filter” refers broadly to devices, materials, and the likethat are able to allow certain components of a mixture to pass throughwhile retaining other components. For example, a filter may comprise amesh with pores sized to allow a blood product (e.g. red blood cellcomposition) to pass through, while retaining other components such asresin particles. The term “filter” is not limited to the means by whichcertain components are retained.

The term “heterocyclic compounds” refers broadly to cyclic compoundswherein one or more of the rings contains more than one type of atom. Ingeneral, carbon represents the predominant atom, while the other atomsinclude, for example, nitrogen, sulfur, and oxygen. Examples ofheterocyclic compounds include furan, pyrrole, thiophene, and pyridine.

The phrase “high temperature activation process” refers to a hightemperature process that typically results in changes in surface area,porosity and surface chemistry of the treated material due to pyrolysisand/or oxidation of the starting material.

The term “Immobilized Adsorbent Device (IAD)” refers to immobilizedadsorbent material entrapped in or attached to an inert matrix. Wherethe inert matrix is a fiber network the term IAD can be usedinterchangeably with the term fiberized resin. For example, fiberizedAmbersorb 572 and Ambersorb IAD (AQF) refer to the same material.

The term “inert matrix” refers to any synthetic or naturally occurringfiber or polymeric material which can be used to immobilize adsorbentparticles without substantially affecting the desired biologicalactivity of the blood product. The matrix may contribute to thereduction in concentration of small organic compounds although typicallyit does not contribute substantially to the adsorption or removalprocess. In addition, the inert matrix may interact with cellular orprotein components resulting in cell removal (e.g. leukodepletion) orremoval of protein or other molecules.

The term “isolating” refers to separating a substance out of a mixturecontaining more than one component. For example, platelets may beseparated from whole blood. The product that is isolated does notnecessarily refer to the complete separation of that product from othercomponents.

The term “macropores” generally means that the diameter of the pores isgreater than about 500 Å. The term micropores refers to pores withdiameters less than about 20 Å. The term mesopores refers to pores withdiameters greater than about 20 Å. and less than about 500 Å.

The term “macroporous” is used to describe a porous structure having asubstantial number of pores with diameters greater than about 500 Å.

The term “macroreticular” is a relative term that means that thestructure has a high physical porosity (i.e., a large number of poresare present) a porous adsorbent structure possessing both macropores andmicropores.

The term “mesh enclosure,” “mesh pouch” and the like refer to anenclosure, pouch, bag or the like manufactured to contain multipleopenings. For example, the present invention contemplates a pouch,containing the immobilized adsorbent particle, with openings of a sizethat allow a blood product to contact the immobilized adsorbentparticle, but retain the immobilized adsorbent particle within thepouch.

The term “partition” refers to any type of device or element that canseparate or divide a whole into sections or parts. For example, thepresent invention contemplates the use of a partition to divide a bloodbag, adapted to contain a blood product, into two parts. The bloodproduct occupies one part of the bag prior to and during treatment,while the adsorbent resin occupies the other part. In one embodiment,after treatment of the blood product, the partition is removed (e.g.,the integrity of the partition is altered), thereby allowing the treatedblood product to come in contact with the adsorbent resin. The partitionmay either be positioned in the bag's interior or on its exterior. Whenused with the term “partition,” the term “removed” means that theisolation of the two parts of the blood bag no longer exists; it doesnot necessarily mean that the partition is no longer associated with thebag in some way.

The term “photoproduct” refers to products that result from thephotochemical reaction that a psoralen or other dyes (e.g., methyleneblue, phthalocyanine) undergo upon exposure to ultraviolet radiation.

The term “polyaromatic compounds” refers to polymeric compoundscontaining aromatic groups in the backbone, such as polyethyleneterphalate, or as pendant groups, such as polystyrene, or both.

The term “polystyrene network” refers broadly to polymers containingstyrene (C₆H₅CH═CH₂) monomers; the polymers may be linear, consisting ofa single covalent alkane chain with phenyl substituents, orcross-linked, generally with m- or p-phenylene residues or otherbifunctional or hypercrosslinked structure, to form a two-dimensionalpolymer backbone or 3D network.

The term “psoralen removal means” refers to a substance or device thatis able to remove greater than about 80% of the psoralen from, e.g., ablood product; preferably, greater than about 90%; most preferablygreater than about 99%. A psoralen removal means may also remove othercomponents of the blood product, such as psoralen photoproducts.

The phrase “reducing the concentration” refers to the removal of someportion of low molecular weight compounds from a biological composition.While reduction in concentration is preferably on the order of greaterthan about 70%, more preferably on the order of about 90%, and mostpreferably on the order of about 99%.

The phrase “removing substantially all of said portion of a compound(e.g. a psoralen, psoralen derivative, isopsoralen, acridine, acridinederivative, dye, plasticizer or activated complement) free in solution”refers preferably to the removal of more than about 80% of the compoundfree in solution, more preferably to the removal of more than about 85%,even more preferably of more than about 90%, and most preferably to theremoval of more than about 99%.

The term “resin” refers to a solid support (such as particles or beadsetc.) capable of interacting and adsorbing to various small organiccompounds, including psoralens, in a solution or fluid (e.g., a bloodproduct), thereby decreasing the concentration of those elements insolution. The removal process is not limited to any particularmechanism. For example, a psoralen may be removed by hydrophobic orionic interaction. The term “adsorbent resin” refers broadly to bothnatural organic substances and synthetic substances and to mixturesthereof.

The term “agitation means” refers to any method by which a biologicalcomposition can be mixed. Examples of agitation means include, withoutlimitation, the following mechanical agitators: reciprocating, orbital,3-D rotator and rotator type agitators.

The term “shaker device” refers to any type of device capable ofthoroughly mixing a blood product like a platelet concentrate. Thedevice may have a timing mechanism to allow mixing to be restricted to aparticular duration.

The term “sintered medium” refers to a structure which is formed byapplying heat and pressure to a porous resin, including for example aparticulate thermoplastic polymer. Porous resins can be prepared bymixing particulate of relatively low melting polymers and heating themso the plastic particles partially fuse but still allow a path forfluids to penetrate the porous mass. Sintered adsorbent media can beprepared similarly by incorporating carbon or other high or non-meltingadsorbent particle with that of the low melting powder and heating.Methods of producing porous plastic materials are described in U.S. Pat.Nos. 3,975,481, 4,110,391, 4,460,530, 4,880,843 and 4,925,880,incorporated by reference herein. The process causes fusing of the lowmelting particles resulting in the formation of a porous solidstructure. The sintered medium can be formed into a variety of shapes byplacing the polymer particles in a forming tool during the sinteringprocess. Adsorbent particles can be introduced into the sintered mediumby mixing adsorbent particles with the thermoplastic polymer particlesbefore subjecting to the sintering process.

The term “stabilizing agent” refers to a compound or composition capableof maintaining the adsorption capacity of certain adsorbents (e.g.,Amberlites) under drying conditions. Generally speaking, acceptablestabilizing agents should be soluble in water and ethanol (or otherwetting agents), nonvolatile relative to water and ethanol, and safe fortransfusion in small amounts. Examples of stabilizing agents include,but are not limited to, glycerol and low molecular weight PEGs. A“wetting agent” is distinguishable from a “stabilizing agent” in thatthe former is believed to reopen adsorbent pores of those resins thatare not hypercrosslinked (e.g., Amberlite XAD-4, Amberlite XAD-16).Wetting agents generally will not prevent pores from collapsing underdrying conditions, whereas stabilizing agents will. A general discussionof wetting and wetting agents is set forth in U.S. Pat. No. 5,501,795 toPall et al., hereby incorporated by reference.

The phrase “substantially maintaining a desired biological activity ofthe biological composition” refers to substantially maintainingproperties (e.g., cellular integrity) of the biological composition. Insome embodiments, the cellular integrity is reflective of the potentialperformance of the composition in a therapeutic setting. For example,where red blood cells are concerned, in vivo activity is not destroyedor significantly lowered if ATP levels, extracellular potassium leakage,% hemolysis are substantially maintained in red blood cells treated bythe methods described herein. For example, the change in ATP level ofthe treated red blood cells should be less than about 10%. The hemolysislevel in the treated red blood cells following storage should be lessthan about 1%, preferably less than about 0.8%. The change inextracellular potassium leakage of the treated red blood cells should beless than about 15%. Where platelets are concerned, in vivo activity isnot destroyed or significantly lowered if, for example, platelet yield,pH, aggregation response, shape change, GMP-140, morphology or hypotonicshock response are substantially maintained in platelets treated by themethods described herein. For example, platelet loss in a biologicalcomposition after storage is preferably less than 15%; more preferably15% after 5 days storage; even more preferably 10% after 5 days storage.It is further contemplated that the phrase substantially maintained foreach of the properties associated with a described blood products mayalso include values acceptable to those of ordinary skill in the art asdescribed in the literature, including for example in Klein H. G., ed.Standards for Blood Banks and Transfusion Services, 17^(th) Ed., aBethesda, Md.: American Association of Blood Banks, 1996, incorporatedby reference herein.

The term “equivalent thereto” when used in reference to a device of thepresent invention refers to a device that functions equivalently withrespect to the maintenance of biological activity of a biologicalcomposition. For example, an “equivalent” device or matrix containingadsorbent particles is one that similarly maintains cell viability or asuitable coagulation factor level.

The term “low molecular weight compound” refers to an organic orbiological molecule having a molecular weight ranging from about 100g/mol to about 30,000 g/mol. Low molecular weight compounds include,without limitation, the following compounds: small organic compoundssuch as psoralens, acridines or dyes; quenchers, such as glutathione;plastic extractables, such as plasticizers; biological modifiers, suchas activated complement, that possess a molecular weight between about100 g/mol and about 30,000 g/mol; and, polyamine derivatives.

The term “biological composition that is suitable for infusion” refersto a biological composition that maintains its essential biologicalproperties (e.g. platelet morphology) while having sufficiently lowlevels of any undesired compounds (e.g. inactivation compounds, responsemodifiers) such that infusion provides intended function withoutdetrimental side effects.

The term “control,” as used in phrases such as “relative to control,”refers to an experiment performed to study the relative effects ofdifferent conditions. For example, where a biological composition istreated with a device, “untreated control” would refer to the biologicalcomposition treated under the same conditions except for the absence oftreatment with the device, or treated with an alternative form of adevice (e.g., immobilized particles vs. non-immobilized particles).

The term “4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen” isalternatively referred to as “S-59.”

The term “N-(9-acridinyl)-β-alanine” is alternatively referred to as“5-[(β-carboxyethyl)amino]acridine.” It is further alternativelyreferred to as “S-300.”

The term “XUS-43493” is alternatively referred to as “Optipore 493.”

Adsorbent Particles

Provided are adsorbent particles which are useful in a device forreducing the concentration of compounds in a biological compositioncontaining cells while substantially maintaining a desired biologicalactivity of the biological composition. Typically, the compounds thatare reduced in the biological composition have molecular weights rangingfrom about 100 g/mol to about 30,000 g/mol.

The adsorbent particles can be of any regular or irregular shape thatlends itself to incorporation into the inert matrix but are preferablyroughly spherical. The particles are greater than about 100 μm indiameter and less than about 1500 μm in diameter; preferably, theparticles are between about 200 μm and about 1300 μm in diameter; morepreferably, the particles are between about 300 μm and about 900 μm indiameter.

A high surface area is characteristic of the particles. Preferably, theparticles have a surface area between about 750 m²/g and about 3000m²/g. More preferably, the particles have a surface area between about1000 m²/g and about 3000 m²/g.

Adsorbent particles suitable for use in the device of the presentinvention can be any suitable material, with the limitation that thematerial does not substantially adversely affect the biological activityof a biological composition upon contact. The adsorbent particle can be,for example, made of materials such as activated carbon, hydrophobicresins or ion exchange resins.

In one preferred embodiment the adsorbent particles are activatedcarbons derived either from natural or synthetic sources. Preferably theactivated carbons are derived from synthetic sources. Nonlimitingexamples of activated carbons include; Picatiff Medicinal®, which isavailable from PICA USA Inc. (Columbus, Ohio), Norit® ROX 0.8, which isavailable from Norit Americas, Inc. (Atlanta, Ga.), Ambersorb® 572,which is available from Rohm & Haas (Philadelphia, Pa.), and G-277®,which is available from PICA (Columbus, Ohio).

In another preferred embodiment, the particles can be hydrophobicresins. Nonlimiting examples of hydrophobic resins include the followingpolyaromatic adsorbents: Amberlite® adsorbents (e.g., Amberlite® XAD-2,XAD-4, and XAD-16), available from Rohm and Haas (Philadelphia, Pa.);Amberchrom® adsorbents available from Toso Haas (TosoHass,Montgomeryville, Pa.); Diaion®//Sepabeads® Adsorbents (e.g., Diaion®HP20), available from Mitsubishi Chemical America, Inc. (White Plains,N.Y.); Hypersol-Macronet® Sorbent Resins (e.g., Hypersol-Macronet®Sorbent Resins MN-200, MN-150 and MN-400) available from Purolite (BalaCynwyd, Pa.); and Dowex® Adsorbents (e.g., Dowex® XUS-40323, XUS-43493,and XUS-40285), available from Dow Chemical Company (Midland, Mich.).

Preferred particles are hydrophobic resins which are polyaromaticadsorbents comprising a hypercrosslinked polystyrene network, such asDowex® XUS-43493 (known commercially as Optipore® L493 or V493) andPurolite MN-200.

Hypercrosslinked polystyrene networks, such as Dowex® XUS-43493 andPurolite MN-200 are non-ionic macroporous and macroreticular resins. Thenon-ionic macroreticular and macroporous Dowex® XUS-43493 has a highaffinity for psoralens, including for example,4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen, and it possessessuperior wetting properties. The phrase “superior wetting properties”means that dry (i.e. essentially anhydrous) adsorbent does not need tobe wet with a wetting agent (e.g., ethanol) prior to being contactedwith the blood product in order for the adsorbent to effectively reducethe concentration of small organic compounds from the blood product.

Hypercrosslinked polystyrene networks, such as Dowex® XUS-43493 andPurolite MN-200 are preferably in the form of spherical particles with adiameter range of about 200 μm to about 1300 μm. Adsorbent particles,including for example, Dowex® XUS-43493, preferably have extremely highinternal surface areas and relatively small pores (e.g. average diameter46 Å). The internal surface area of the particle can be from about 300to about 1100 m²/g; preferably about 900 to about 1100 m²/g; mostpreferably about 1100 m²/g. The majority of the pores of the particlecan be greater than 25 Å and less than 800 Å; preferably from about 25 Åto about 150 Å; most preferably from about 25 Å to about 50 Å. While itis not intended that the present invention be limited to the mechanismby which reduction of small organic compounds takes place, hydrophobicinteraction is believed to be the primary mechanism of adsorption. Itsporous nature confers selectively on the adsorption process by allowingsmall molecules to access a greater proportion of the surface arearelative to large molecules (i.e., large proteins) and cells. Purolite®has many similar characteristics to Dowex® XUS-43493, such as highaffinity for psoralens and superior wetting properties, and is also apreferred adsorbent particle.

Polystyrene particles can be classified, based on their mechanism ofsynthesis and physical and functional characteristics, as i)conventional networks and ii) hypercrosslinked networks. Preferredadsorbents have a high surface area, have pores that do not collapseupon drying, do not require wetting for biological compositionscomprising red blood cells or platelets, and have extremely low levelsof small particles and foreign particles (e.g. dust, fibers,non-adsorbent particles, and unidentified particles). In addition,preferred adsorbents have low levels of extractable residual monomer,crosslinkers and other organic extractables.

The conventional networks are primarily styrene-divinylbenzenecopolymers in which divinylbenzene (DVB) serves as the crosslinkingagent (i.e., the agent that links linear polystyrene chains together).These polymeric networks include the “gel-type” polymers. The gel-typepolymers are homogeneous, non-porous styrene-DVB copolymers obtained bycopolymerization of monomers. The macroporous adsorbents represent asecond class of conventional networks. They are obtained bycopolymerization of monomers in the presence of diluents thatprecipitate the growing polystyrene chains. The polystyrene networkformed by this procedure possess a relatively large internal surfacearea (up to hundreds of square meters per gram of polymer); Amberlite®XAD-4 is produced by such a procedure.

In contrast to the conventional networks described above, the preferredadsorbents of the present invention (e.g., Dowex® XUS-43493) arehypercrosslinked networks. These networks are produced by crosslinkinglinear polystyrene chains either in solution or in a swollen state withbifunctional agents; the preferred bifunctional agents produceconformationally-restricted crosslinking bridges, that are believed toprevent the pores from collapsing when the adsorbent is in anessentially anhydrous (i.e., “dry”) state.

The hypercrosslinked networks are believed to possess three primarycharacteristics that distinguish them from the conventional networks.First, there is a low density of polymer chains because of the bridgesthat hold the polystyrene chains apart. As a result, the adsorbentsgenerally have a relatively large porous surface area and pore diameter.Second, the networks are able to swell; that is, the volume of thepolymer phase increases when it contacts organic molecules. Finally, thehypercrosslinked polymers are “strained” when in the dry state; that is,the rigidity of the network in the dry state prevents chain-to-chainattractions. However, the strains relax when the adsorbent is wetted,which increases the network's ability to swell in liquid media. Davankovand Tsyurupa, Reactive Polymers 13:27-42 (1990); Tsyurupa et al.,Reactive Polymers 25:69-78 (1995), hereby incorporated by reference.

Several cross-linking agents have been successfully employed to producethe bridges between polystyrene chains, including p-xylene dichloride(XDC), monochlorodimethyl ether (MCDE), 1,4-bis-chloromethyldiphenyl(CMDP), 4,4′-bis-(chloromethyl)biphenyl (CMB), dimethylformal (DMF),p,p′-bis-chloromethyl-1,4-diphenylbutane (DPB), andtris-(chloromethyl)-mesitylene (CMM). The bridges are formed betweenpolystyrene chains by reacting one of these cross-linking agents withthe styrene phenyl rings by means of a Friedel-Crafts reaction. Thus,the resulting bridges link styrene phenol rings present on two differentpolystyrene chains. See, e.g., U.S. Pat. No. 3,729,457, herebyincorporated by reference.

The bridges are especially important because they generally eliminatethe need for a “wetting” agent. That is, the bridges prevent the poresfrom collapsing when the adsorbent is in an essentially anhydrous (i.e.,“dry”) state, and thus they do not have to be “reopened” with a wettingagent prior to the adsorbent being contacted with a blood product. Inorder to prevent the pores from collapsing, conformationally-restrictedbridges should be formed. Some bifunctional agents like DPB do notresult in generally limited conformation; for example, DPB contains foursuccessive methylene units that are susceptible to conformationrearrangements. Thus, DPB is not a preferred bifunctional agent for usewith the present invention.

Some of the structurally-related characteristics of the above-describedadsorbent particles are summarized in Table A.

TABLE A Mean Surface Mean Pore Mesh Resin Chemical Nature Area (m²/g)Diam. (Å) Size (μm) Amberlite ® Adsorbents - Rohm and Haas XAD-2polyaromatic 300 90 20-60 XAD-4 polyaromatic 725 40 20-60 XAD-7polymethacrylate 450 90 20-60 XAD-16 polyaromatic 800 100 20-60 XAD-1180polyaromatic 600 300 20-60 XAD-2000 polyaromatic 580 42 20-60 XAD-2010polyaromatic 660 280 20-60 Amberchrom ® Adsorbents - Toso Haas CG-71mpolymethacrylate 450-550 200-300  50-100 CG-71c polymethacrylate 450-550200-300  80-160 CG-161m polyaromatic 800-950 110-175  50-100 CG-161cpolyaromatic 800-950 110-175  80-160 Diaion ®//Sepabeads ® Adsorbents -Mitsubishi Chemical HP20 polyaromatic 500 300-600 20-60 SP206 brominated550 200-800 20-60 styrenic SP207 brominated 650 100-300 20-60 styrenicSP850 polyaromatic 1000  50-100 20-60 HP2MG polymethacrylate 500 200-80025-50 HP20SS polyaromatic 500 300-600  75-150 SP20MS polyaromatic 500300-600  50-100 Dowex ® Adsorbents - Dow Chemical Company XUS-40285functionalized 800 25 20-50 XUS-40323 polyaromatic 650 100 16-50XUS-43493 polyaromatic 1100 46 20-50

Processing the Adsorbent Particles

The adsorbent particles may be further processed to remove fineparticles, salts, potential extractables, and endotoxin. The removal ofthese extractable components is typically performed by treatment witheither organic solvents, steam, or supercritical fluids. Preferably theparticles are sterilized.

Several companies currently sell “cleaned” (i.e., processed) versions ofcommercially available adsorbent particles. In addition to processingthe adsorbent particles (e.g. resins), these companies test theadsorbents, and the final adsorbent is certified sterile (USP XXI),pyrogen-free (LAL), and free of detectable extractables (DVB and totalorganics).

Thermal processing (e.g., steam) is an effective method for processingadsorbent particles. F. Rodriguez, Principles Of Polymer Systems,(Hemisphere Publishing Corp.), pp. 449-53 (3rd. Ed., 1989). Supelco,Inc. (Bellefonte, Pa.) uses a non-solvent, thermal proprietary processto clean the Dowex® XUS-43493 and Amberlite adsorbents. The mainadvantage of using steam is that it does not add any potentialextractables to the adsorbent. One big disadvantage, however, is thatthis process can strip water from the pores of the resin beads;effective performance of some adsorbents requires that the beads bere-wet prior to contacting the blood product.

One advantage of the cleaned/processed adsorbent is an extremely lowlevel of particles with diameters less than 30 μm. Preliminary testingon adsorbents (Dowex® XUS-43493 and Amberlite® XAD-16) processed bySupelco was performed to determine particle counts. The results of thesetests indicated that foreign particles (e.g., dust, fibers,non-adsorbent particles, and unidentified particles) were absent andthat fine particles (<30 μm) were essentially absent.

The Use of Wetting Agents and Stabilizing Agents with Adsorbent Resins

Methods may be used for preventing drying and loss of adsorptioncapacity of particles, such as Amberlite® which lose some of theiradsorption capacity under certain conditions (e.g., drying).

In one method, particles, materials or devices may be manufactured in awet state which is sealed and not capable of drying. This method isassociated with several important drawbacks. The shelf-life of theproducts could be reduced since levels of extractables from thematerials could increase over time. Sterilization may be limited to asteam process because γ-irradiation of wet polymers is typically notperformed. Manufacturing a device that requires that a component bemaintained in a wet state is, in general, more difficult thanmanufacturing a dry device; for example, bioburden and endotoxin maybecome of concern if there is a long lag time between device assemblyand terminal sterilization.

A second method for preventing loss of adsorption capacity involves theuse of an adsorbent which is not adversely affected by drying. Aspreviously set forth, macroreticular adsorbents possessing highlycrosslinked porous structures (e.g., Dowex® XUS-43493 and Purolite®MN-200) generally do not require a wetting agent because the crosslinksprevent the pores from collapsing. Unlike Amberlite® XAD-16, thesemacroreticular adsorbents retain a very high proportion of their initialactivity when they are dried.

In a third method, loss of adsorption capacity upon drying may beprevented by hydrating Amberlite® XAD-16 and related adsorbents (e.g.,Amberlite® XAD-4) in the presence of a non-volatile wetting agent. Forexample, when using Amberlite® XAD-16 as the adsorbent, the adsorbentbeads may partially dry prior to use during handling, sterilization, andstorage. When the water content of these adsorbents drops below acritical level, a rapid loss in adsorption capacity occurs (probably dueto “collapse” of the pores); thus, for optimum effectiveness, the poreshave to be “reopened” with a wetting agent prior to use.

Stabilizing agents are effective in maintaining adsorption capacity nearits maximum when certain adsorbent resins are subjected to dryingconditions. It is believed that the use of stabilizing agents serves toprevent the adsorbent pores from collapsing.

An acceptable stabilizing agent should be soluble in water and ethanol,nonvolatile relative to ethanol and water, and safe for transfusion insmall amounts. Glycerol and low molecular weight polyethylene glycol(e.g., PEG-200 and PEG-400) are examples of stabilizing agentspossessing these characteristics. Glycerol has a positivehemocompatibility history. It is frequently added to blood as acryo-preservative agent in the frozen storage of red blood cellpreparations. See, e.g., Chaplin et al., Transfusion 26:341-45 (1986);Valeri et al., Am. J. Vet. Res. 42(9)1590-94 (1981). Solutionscontaining up to 1% glycerol are routinely transfused, and glycerolsolutions are commercially available (e.g., Glyerolite 57 Solution,Fenwal Laboratories, Deerfield, Ill.). Adsorbent beads like Amberlite®XAD-16 may be stabilized in ethanol and glycerol.

Low molecular weight polyethylene glycols, commonly used aspharmaceutical bases, may also be used as stabilizing agents. PEGs areliquid and solid polymers of the general chemical formulaH(OCH₂CH₂)_(n)OH, where n is greater than or equal to 4. PEGformulations are usually followed by a number that corresponds to itsaverage molecular weight; for example, PEG-200 has a molecular weight of200 and a molecular weight range of 190-210. PEGs are commerciallyavailable in a number of formulations (e.g., Carbowax, Poly-G, andSolbase).

Inert Matrices for Particle Immobilization

The adsorbent particles are immobilized by an inert matrix. The inertmatrix can be made of a synthetic or natural polymer. For example, theinert matrix can be a synthetic or natural polymer fiber, for example, afiber network. The inert matrix can be sintered polymers. The inertmatrix, as with the other components of the device, preferably isbiocompatible and does not substantially adversely affect the biologicalactivity of a material upon contact.

Most preferably, the synthetic fibers are polyester fibers (Air QualityFiltration (AQF), a division of Hoechst Celanese (Charlotte, N.C.)).Other preferred examples of synthetic fibers are polyethylene orpolyamide fibers. Other exemplary synthetic fibers include polyolefin,polyvinyl alcohol and polysulfone fibers.

In a preferred embodiment, the synthetic polymer fiber includes a firstpolymer core with a high melting point surrounded by a sheath with alower melting temperature. The polymer core can be a polyester(polyethylene terephthalate). The sheath can be a nylon, or a modifiedpolyester. Fibers are commercially available from Unitika (Osaka, Japan)and Hoechst Trevira GmbH & Co. (Augsberg, Germany).

Exemplary natural polymer fibers include cellulose fibers derived from avariety of sources, such as jute, kozu, kraft and manila hemp. Networksof synthetic or natural polymer fibers have been used to make filters asdescribed in U.S. Pat. Nos. 4,559,145 and 5,639,376, which are hereinincorporated by reference.

Synthetic polymers suitable for the construction of sintered particlesare high density polyethylene, ultra high molecular weight polyethylene,polypropylene, polyvinyl fluoride, polytetrafluoroethylene, nylon 6.More preferably the sintered particles are polyolefins, such aspolyethylene.

Polymeric fibers such as those described above may be adsorbent resinswithout the attachment of adsorbent particles. Such fibers may be formedinto a fiber network or may be immobilized on a fiber network of a lessadsorbent fiber. Such fibers are contemplated by the present invention;such fibers preferably contain a large, porous, adsorptive surface areaor other adsorptive means to facilitate reduction in the concentrationof low molecular weight compounds.

Immobilization of Particles

In one embodiment, the adsorbent particles are immobilized by an inertmatrix to produce an adsorption medium for reducing the concentration ofsmall organic compounds in a material. The inert matrix can be a threedimensional network including a synthetic or natural polymer fibernetwork with adsorbent particles immobilized therein.

Preferably, the adsorption medium comprises small porous adsorbentparticles with highly porous structures and very high internal surfaceareas, as described above, immobilized by the inert matrix. Preferably,when a biological material is brought into contact with the adsorptionmedium, the adsorption medium does not substantially adversely affectthe biological activity or other properties of the material.

Technology for immobilization of adsorbent beads on a fiber network toconstruct air filters has been described in U.S. Pat. No. 5,486,410 andU.S. Pat. No. 5,605,746, incorporated by reference herein. As depictedin FIG. 1, the polymer fibers 600 of the fiber network consist of apolymer core 602 (e.g., polyethylene terephthalates (PET)) with a highmelting point surrounded by a polymer sheath 604 (e.g., nylon) with arelatively low melting temperature. See U.S. Pat. No. 5,190,657 toHeagle et al., hereby incorporated by reference. The fiberized resin isprepared by first evenly distributing the adsorbent beads in the fibernetwork. Next, the network is rapidly heated (e.g., 180° C.×1 min.)causing the polymer sheath of the fibers 600 to melt and bond to theadsorbent beads 606 and other fibers, forming a cross-linked fibernetwork, represented in FIG. 2. As depicted in FIG. 3 and FIG. 4 (not toscale), generally speaking, the fiber networks contain three layers; twoouter layers 607 that are densely packed with fibers 600 and a lessdense inner layer 609 that contains the adsorbent beads 606 and fewerfibers 600. In a preferred embodiment, the edges of the fiberized resinmay be sealed with polyurethane or other polymers. Alternatively, asdepicted in FIG. 3 and FIG. 4, heat seals 608 may be made in theresulting fiberized resin at predetermined intervals; for example, heatseals can be made in the fiberized resin in a pattern of squares.Thereafter, the fiberized resin can be cut through the heat seals toform samples of resin containing a desired mass (e.g., preferably lessthan 5.0 g and more preferably less than 3.0 g) of adsorbent beads andof a size suitable for placement within a blood product container. Theheat seals serve to prevent the cut fiberized resin from fraying andhelp to immobilize the adsorbent beads. However, the use of such heatseals is not required in order to practice the present invention. In analternative embodiment, depicted in FIG. 4, the adsorbent beads 606 arenot secured to the fibers themselves, but rather are immobilized betweenthe denser outer layers 607 of fibers and with the heat seals 608; thisembodiment may also result in samples of fiberized media containing adefined amount of adsorbent after being cut through the heat seals.

The present invention also contemplates the use of an adhesive (e.g., abonding agent) to secure the adsorbent resin to the fibers. Moreover,while it is preferable that the adsorbent beads be chemically attachedto the fiber network, the beads may also be physically trapped withinthe fiber network; this might be accomplished, for example, bysurrounding the beads with enough fibers so as to hold the beads inposition.

Other ways that the adsorbent particles may be immobilized in a fibernetwork are also contemplated. The particles can be immobilized using adry-laid process, as described in U.S. Pat. Nos. 5,605,746 and 5,486,410(AQF patents), which are herein incorporated by reference. The particlescan be immobilized using a wet-laid process, as described in U.S. Pat.Nos. 4,559,145 and 4,309,247, which are herein incorporated byreference. The particles can be immobilized using a melt-blown process,as described in U.S. Pat. No. 5,616,254, which is herein incorporated byreference. Where a wet-laid process is used to construct a matrix fromnatural polymer fibers, the inert matrix preferably includes a bindingagent to bond the adsorbent particles to the fibers. Nonlimitingexamples of binding agents include melamine, polyamines and polyamides.The matrix typically contains 1% or less of such binding agents.

Where the inert matrix is constructed from particles of syntheticpolymers which are sintered with adsorbent particles, it is importantthat the adsorbent particle have a higher melting temperature than thematrix.

In a preferred embodiment, the adsorbent particles are immobilized in afiber matrix that is formed by thermal bonding of a biocomponent fibernetwork. An alternative embodiment involves immobilizing adsorbentparticles in non-biocomponent fibers and using a wet strength resinsystem, adhesives or additional fusible fibers to form bonds between thefibers and adsorbent particles. Nonlimiting examples of useful fibersinclude polyester, nylon and polyolefin. (Suppliers of fibers for thenonwovens industry have been listed in “A Guide to Fibers forNonwovens,” Nonwovens Industry, June 1998, 66-87.) Examples of wetstrength resin systems include melamine/formaldehyde,epichlorohydrin-based resins, polyamines and polyamides. The use of heatfusible fibers for immobilizing particles in fiber matrices has beendisclosed. See, e.g., U.S. Pat. No. 4,160,059.

Preferably, the resulting adsorption medium comprises known amounts ofadsorbent per area. The adsorbent per area is from about 100 g/m² toabout 500 g/m², preferably from about 250 g/m² to about 350 g/m². Thus,the appropriate amount of adsorbent contemplated for a specific purposecan be measured simply by cutting a predetermined area of the fiberizedresin (i.e., there is no weighing of the fiberized resin).

The adsorption medium preferably is biocompatible (i.e., not producing atoxic, injurious, or immunologic response); has a minimal impact on theproperties of the material such as blood product (e.g., platelets andclotting factors); and is not associated with toxic extractables. Theimmobilized adsorbent particles of the adsorption medium preferably havehigh mechanical stability (i.e., no fine particle generation). Theadsorption medium for a batch device contains about 25-85% adsorbent byweight, preferably about 50-80% adsorbent at a loading of about 100-500g/m², more preferably about 50-80% adsorbent at a loading of about250-350 g/m².

Coating the Adsorbent Particles

The surface hemocompatibility of the particles, matrices or adsorptionmedium can be improved by coating their surfaces with a hydrophilicpolymer. Exemplary hydrophilic polymers include poly(2-hydroxyethylmethacrylate) (pHEMA), which may be obtained from, e.g., ScientificPolymer Products, Inc. (Ontario, N.Y.) and cellulose-based polymers,e.g., ethyl cellulose, which may be obtained from Dow Chemical (Midland,Mich.). See, e.g., Andrade et al., Trans. Amer. Soc. Anil: Int. OrgansXVII:222-28 (1971). Other examples of coatings include polyethyleneglycol and polyethylene oxide, also available from Scientific PolymerProducts, Inc. The polymer coating can increase hemocompatibility andreduce the risk of small particle generation due to mechanicalbreakdown.

The adsorbent surface may also be modified with immobilized heparin. Inaddition, strong anion exchange polystyrene divinylbenzene adsorbentsmay be modified via heparin adsorption. Heparin, a polyanion, willadsorb very strongly to the surfaces of adsorbents which have stronganion exchange characteristics. A variety of quaternary amine-modifiedpolystyrene divinyl benzene adsorbents are commercially available.

The coating can be applied in a number of different methods, includingradio frequency glow discharge polymerization, as described in U.S. Pat.No. 5,455,040, which is hereby incorporated by reference and the Wurstercoating process (performed by International Processing Corp.(Winchester, Ky.).

In one embodiment, the Wurster coating process can be applied bysuspending the adsorbent particles (generally via air pressure) in achamber such that the hydrophilic polymer, such as pHEMA, can be sprayedevenly onto all surfaces of the adsorbent particle. As illustrated inExample 3, Dowex® XUS-43493 sprayed evenly with pHEMA demonstrated anincrease in platelet yield as well as a dramatic effect on plateletshape change with increasing amounts of coating. It was found that theWurster coating process selectively coated the outside surface of theadsorbent surface, leaving the inside porous surface nearly unaffected.

In a preferred embodiment, the coating can be applied by soaking theimmobilized adsorption medium in the hydrophilic polymer (see Example3). This process is simpler and less expensive than spraying theadsorbent particles with the hydrophilic polymer.

The process is not limited to a process that applies the coating of theadsorption medium at any particular time. For example, in oneembodiment, the pHEMA coating is applied after production of theadsorption medium, but prior to heat sealing the adsorption medium. Inanother embodiment, the adsorption medium is first heat sealed, and thenthe pHEMA coating is applied. In addition to coating the adsorptionmedium, the rinsing process associated with pHEMA application serves toremove loose particles and fibers.

As the amount of coating is increased, it becomes more difficult forsome small organic compounds to cross the coating to reach the particlesurface, resulting in a decrease in adsorption kinetics. Thus, as theamount of coating is increased, an increased mass of adsorbent mustgenerally be used to achieve the same removal kinetics as coatedadsorbent. In one embodiment, the optimum level of pHEMA coating is theminimum coating at which a protective effect on platelet yield and invitro platelet function is observed (0.1-0.5%).

The coatings may be sensitive to sterilization. For example, gammasterilization may result in cross-linking and/or scission of thecoating. Therefore, the type (E-beam vs. gamma irradiation) and dose ofsterilization may influence the properties of the coated adsorbent.Generally, E-beam sterilization is preferred.

Devices

Devices are provided for reduction of compounds from biologicalcompositions containing cells. The device is a batch device. An exampleof a batch device is shown in FIG. 16. Batch devices are known in theliterature and are described, for example, in PCT publication WO96/40857, incorporated by reference herein.

Batch devices of the invention may comprise a container, such as a bloodbag, including the adsorbent medium containing immobilized particles. Inone embodiment a blood product is added to a blood bag containing theadsorbent medium and the bag is agitated for a specified period of time.

For example, in one embodiment, an adsorption medium, e.g., immobilizedDowex® XUS-43493, is placed inside a blood product container (e.g., a PL146 Plastic container (BaxterHealthcare Corp. (Deerfield, Ill.)) kept oneither a platelet shaker (Helmer Laboratories (Novesvill, Ind.)) orrotator (Helmer Laboratories (Novesvill, Ind.)) for about 24 hours atroom temperature and stored under various temperature conditions. Thesize of the blood product container can be from about 600 to about 1200mL. The storage temperature can be from about 4° C. to about 22° C.

Methods can be used to reduce the presence of adsorbent particles thatmay come loose from the adsorbent medium.

The present invention contemplates a batch device including theimmobilized adsorbent medium retained in a container such as a meshbag/pouch. The mesh/pouch can be constructed of a woven, non-woven ormembranous enclosure. In one embodiment, the woven mesh pouch can beconstructed of medical-grade polyester or nylon. The preferredembodiment is polyester. Commercially-available membranes include, butare not limited to, Supor® 200, 800, 1200 hydrophilic polyethylenesulfonate (PES) membranes (Gelman Sciences (Ann Arbor, Mich.));Durapore® hydrophilic modified polyvinylidene difluoride (PVDF) (ManteeAmerica Corp. (San Diego, Calif.)) and hydrophilic modified polysulfonemembranes with integrated hydrophobic vents, e.g. Gemini membranes,(Millipore (Marlborough, Mass.)); and membranes comprising polycarbonatewith a polyvinylidene coating (Poretics (Livermore, Calif.)). Thecontainers can be sterilized after addition of the adsorption medium.

Preferred Embodiment for Biological Compositions Containing Cells

The present invention provides devices for reducing the concentration oflow molecular weight compounds in biological compositions containingcells. The devices comprise an adsorption medium, which is comprised ofparticles immobilized by an inert matrix.

Biological response modifiers like the anaphalatoxin C3a and theterminal membrane attack complex SC5b-9 have been shown to be producedby the processing, (e.g., leukofiltration, pheresis, recovery of shedblood, etc.) and storage of whole blood and its components. Thesebiological response modifiers have been implicated in adverse events insurgery and transfusion.

In some embodiments, the device of the present invention reduces orcontrols the concentration of activated complement in biologicalcompositions containing cells. The concentration of activated complementin the composition is reduced or controlled when it is treated with thedevice, as opposed to a composition that has not been treated with thedevice. In one embodiment, the adsorption device comprises fiberizedAmbersorb IAD, for example, as produced by AQF. In this embodiment,exposure of biological compositions containing cells to the deviceresults in a reduction in the C3a complement fragment and SC5b-9terminal component complex over control. In one embodiment, exposure tothe device for 5 days results in at least about a 10% reduction of C3acomplement fragment over control. In another embodiment, exposure to thedevice for 5 days results in at least about a 30% reduction of C3acomplement fragment over control. In another embodiment, exposure to thedevice for 5 days results in at least about a 50% reduction of C3acomplement fragment over control.

In one embodiment, the invention provides a device for reducing theconcentration of compounds in a biological composition comprisingplatelets. The biological activity of the platelets is substantiallymaintained after treatment with the device. The adsorption medium ofthis embodiment comprises adsorbent particles immobilized by an inertmatrix. Preferred particles are highly porous and have a surface areagreater than about 750 m²/g.

Particularly preferred particles for this embodiment are polyaromaticadsorbents comprising a hypercrosslinked polystyrene network, such asDowex®XUS-43493 or Purolite MN-200. The preferred inert matrix includesa synthetic or natural polymer fiber. In a preferred embodiment theinert matrix includes a synthetic polymer fiber which includes a firstpolymer core with a high melting point surrounded by a sheath with alower melting temperature. The polymer core can be a polyethyleneterphthalate core. The sheath can be a nylon sheath or a modifiedpolyester sheath. Staple fibers are commercially available from Unitika(Osaka, Japan) and Hoechst Trevira.

Exemplary compounds that are reduced or controlled by the devices,materials and methods of this embodiment are psoralens, psoralenderivatives, isopsoralens, psoralen photoproducts, acridines, acridinederivatives, methylene blue, plastic extractables, biological responsemodifiers, quenchers and polyamine derivatives.

Biological compositions comprising platelets are typically used within 3days of donation but may be stored for up to 7 days at room temperature,therefore, it would be advantageous to allow the platelet compositionsto remain in contact with the adsorption medium for the entire storageperiod. Preferably, the procedure would result in an acceptable plateletyield (e.g., less than 10% loss). One method contemplated by the presentinvention allows extended storage by improving the hemocompatibility ofthe adsorbent surface.

The use of an adsorption medium comprising adsorbent particlesimmobilized by an inert matrix permits the concentration of lowmolecular weight compounds to be reduced without a substantial loss inplatelet count. The phrase “without a substantial loss” refers to aplatelet preparation that is suitable for its intended purpose, forexample, is suitable for infusion into humans, and may refer to, forexample, a loss of platelet count or function of less than about 10%,preferably less than about 5% over a period of time, more preferably atleast 5 days. Furthermore, the time that the platelets may be contactedwith the adsorption medium without substantial loss in platelet count isgreater than the amount of time that the platelets can be contacted withthe adsorbent particles alone. The immobilization of the particlesunexpectedly permits both a longer contact time and a reduction in lossof platelet count. The platelets typically cannot be contacted withnon-immobilized adsorbent particles for more than about 20 hours withouta significant loss of platelet count e.g. about 20% loss. In contrast,platelets may be contacted with the adsorption medium comprisingadsorbent particles immobilized by an inert matrix for more than 20hours, e.g. about 1 to 7 days without a substantial loss in plateletcount.

Additionally in vitro platelet function (e.g., shape change, GMP-140,pH) is improved for platelets stored in the presence of the adsorptionmedium in comparison to the storage of the platelets without theadsorption medium over time. Platelets stored in the presence of theadsorption medium can have a pH from greater than about 6 to less thanabout 7.5.

In another embodiment, the invention provides a device for reducing theconcentration of low molecular weight compounds (e.g., small organiccompounds) in a biological composition comprising red blood cells whilesubstantially maintaining the biological activity of the red bloodcells. Typically, the compounds removed by the device have a molecularweight ranging from about 100 g/mol to about 30,000 g/mol. Theadsorption medium comprises adsorbent particles immobilized by an inertmatrix. Preferred particles for this embodiment are highly porous andhave a surface area greater than about 750 m²/g.

Preferably, a device used for red blood cell compositions is a devicethat substantially maintains the biological activity of the red bloodcells after reduction of the concentration of low molecular weightcompounds. In one embodiment, the red blood cell device does notsubstantially adversely affect the biological activity of a fluid uponcontact. The device embodiment comprises an adsorption medium containingparticles immobilized by an inert matrix and optionally a particleretention device.

In one embodiment the particles used in devices for red blood cellcompositions are activated charcoal. Preferably the activated carbonsare derived from synthetic sources. Nonlimiting examples of activatedcarbons include Picactif Medicinal, which is available from Pica U.S.A.(Columbus, Ohio); Norit ROX 0.8, which is available from Norit AmericasInc. (Atlanta, Ga.); and G-277, which is available from Pica U.S.A.(Columbus, Ohio).

In one embodiment, the adsorbent is preferably an activated carbonderived from a synthetic source, such as Ambersorb 572. The Ambersorbsare synthetic activated carbonaceous (i.e. rich in carbon) adsorbentsthat are manufactured by Rohm & Haas (Philadelphia, Pa.). Ambersorbs aregenerally large spherical (300-900 μm) particles that are more durablethan typical activated carbons. The Ambersorbs are syntheticallymanufactured by treating highly sulfonated porous polystyrene beads witha proprietary high temperature activation process. These adsorbents donot require pre-swelling to achieve optimal adsorption activity.

In another embodiment, the particles used in devices for use withcompositions containing red blood cells (“red blood cell devices”) canbe hydrophobic resins. Nonlimiting examples of hydrophobic resinsinclude the following polyaromatic adsorbents: Amberlite® adsorbents(e.g., Amberlite® XAD-2, XAD-4, and XAD-16), available from Rohm andHaas (Philadelphia, Pa.); Amberchrom® adsorbents available from TosoHaas (Toso Haas, Montgomeryville, Pa.); and Diaion®//Sepabeads®Adsorbents (e.g., Diaion® HP20), available from Mitsubishi ChemicalAmerica, Inc. (White Plains, N.Y.). In a particularly preferredembodiment the particles are Hypersol-Macronet® Sorbent Resins (e.g.,Hypersol-Macronet® Sorbent Resins MN-200, MN-150 and MN-400) availablefrom Purolite (Bala Cynwyd, Pa.) or Dowex® Adsorbents (e.g., Dowex®XUS-43493, and XUS-40285), available from Dow Chemical Company (Midland,Mich.).

The preferred inert matrix of a red blood cell device includes asynthetic or natural polymer fiber. In a preferred embodiment the inertmatrix includes a synthetic polymer fiber which includes a first polymercore with a high melting point surrounded by a sheath with a lowermelting temperature. The polymer core can be a polyethyleneterephthalate or polyester core. The sheath can be a nylon sheath or amodified polyester sheath. Fibers are commercially available fromUnitika (Osaka, Japan) and Hoechst Trevira (Augsberg, Germany).

In some embodiments, the adsorption medium of a red blood cell device isin an enclosure. In one embodiment, the device comprises an adsorptionmedium, and a housing. In another embodiment, the device comprising anadsorption medium and a housing may also include a particle retentionmedium. In one embodiment, the housing comprises a blood bag of a volumebetween about 600 ml and about 1 L. In another embodiment, the housingcomprises a blood bag of a volume between about 800 ml and about 1 L.The particle retention medium may comprise a polyester woven, polyesternon-woven, or synthetic membranous enclosure.

It is preferable that the device contact the red blood composition atabout 4° C. or about 22° C. (room temperature), in the presence ofagitation, over a time period of 1 to 35 days. In one embodiment, thered blood composition is contacted with the device at about 22° C. forno more than about 36 hours. In another embodiment, the red blood cellcomposition is contacted with the device at about 22° C. for no morethan about 24 hours. In another embodiment, the red blood composition iscontacted with the device at about 22° C. for no more than about 12hours. In another embodiment, the red blood composition is contactedwith the device at about 22° C. for no more than 6 hours.

In some embodiments, the temperature is changed after the red bloodcomposition is brought into contact with the device. In one embodiment,the device is contacted with the red blood composition at about 22° C.for a time ranging from about 0.5 to about 24 hours and then stored atabout 4° C. for up to about 5 weeks. In another embodiment, the deviceis contacted with the red blood composition at about 22° C. for a timeranging from about 0.5 to about 12 hours and then stored at about 4° C.for up to about 5 weeks. In another embodiment, the device is contactedwith the red blood composition at about 22° C. for a time ranging fromabout 0.5 to about 6 hours and then stored at about 4° C. for up toabout 5 weeks.

The use of an adsorption medium comprising adsorbent particlesimmobilized by an inert matrix permits the treatment of the red bloodcell composition without a substantial loss in red blood cell function.The phrase “without a substantial loss” refers to a product that wouldbe allowable for transfusion, or its intended purpose, and in someinstances may refer to less than about 1% hemolysis; preferably lessthan about 0.8% hemolysis, greater than about 80% recovery of red bloodcells; preferably greater than 90% recovery of red blood cells, lessthan about 10% difference from no-device control red blood cells inchange in ATP concentration, and less than about 15% difference fromno-device control red blood cells in change in extracellular potassiumconcentration. At 35 days, the change in hemolysis is at least 10% lowerfor the IAD compared to the non-immobilized particles, preferably, 20%lower and more preferably 50%. Most preferably, the change is hemolysisis at least 90% lower for the IAD compared to the non-immobilizedparticles.

Red blood cell function can be assayed using standard kits. Inparticular, hemolysis may be determined by measuring the absorbence at540 nm of a red blood cell supernatant sample in Drabkin's reagent((Sigma Chemical Company), St. Louis, Mo.). Potassium leakage can beassayed using a Na+/K+ analyzer. (Ciba-Corning Diagnostics, Medfield,Mass.). Quantitative enzymatic determination of ATP in total Red BloodCell samples is possible using a standard kit (Sigma Diagnostics, St.Louis, Mo.) and measuring absorbance at 340 nm compared to a waterbackground.

Where the biological composition to be treated is a red blood cellcontaining composition, the device can reduce the concentration of lowmolecular weight compounds in a red blood cell sample. Preferably thedevice can reduce the concentration of both acridine derivatives andthiols in a red blood cell sample. More preferably, the device canreduce the concentration of both 5-[(β-carbethoxyethyl)amino]-acridineand glutathione in a red blood cell sample. Standard HPLC assays can beused to determine concentrations of 5-[(β-carboxyethyl)amino]acridineand glutathione in red blood cells contacted with the device. Assaymobile phases are 10 mM H₃PO₄ in HPLC water and 10 mM H₃PO₄ inacetonitrile. Zorbax SB-CN and YMC ODSAM-303 columns are available fromMacMod Analytical, Inc. (Chadds Ford, Pa.) and YMC, Inc. (Wilmingtion,N.C.).

Where a device is brought into contact with a biological compositioncontaining cells in the presence of agitation, the agitation can beconstant or intermittent. The agitation is provided through any suitablemeans which maintains the functionality of the cells, includingmechanical agitators of the following types: reciprocating, orbital, 3-Drotator and rotator type agitators. In one embodiment, the agitation isprovided by an orbital agitator and is constant. In another embodiment,the agitation is provided by an orbital agitator and is intermittent. Inanother embodiment, the agitation is provided by a reciprocatingagitator.

Applications

The present invention contemplates reducing the concentration of lowmolecular weight compounds from biological compositions containingcells. Such compounds include, for example, pathogen-inactivating agentssuch as photoactivation products, aminoacridines, organic dyes andphenothiazines. Exemplary pathogen inactivating agents includefurocoumarins, such as psoralens and acridines. Following treatment of ablood product with a pathogen inactivating compound as described forexample in U.S. Pat. Nos. 5,459,030 and 5,559,250, incorporated byreference herein, the concentration of pathogen inactivating compoundsin the blood product can be reduced by contacting the treated bloodproduct with a device of the invention.

In one embodiment the present invention contemplates a method ofinactivating pathogens in solution, wherein the method comprises: a)providing, in any order: i) a cyclic compound, ii) a solution suspectedof being contaminated with said pathogens, and iii) fiberized resin; b)treating said solution with said cyclic compound so as to create atreated solution product wherein said pathogens are inactivated; and c)contacting said treated solution product with said fiberized resin, andfurther comprising a device for reducing the concentration of smallorganic compounds in a blood product while substantially maintaining adesired biological activity of the blood product, the device comprisinghighly porous adsorbent particles, wherein the adsorbent particles areimmobilized by an inert matrix.

In addition to the pathogen inactivating compound, reactive degradationproducts thereof can be reduced from the material such as a bloodproduct, for example prior to transfusion.

The materials and devices disclosed herein can be used in apheresismethods. Whole blood can be separated into two or more specificcomponents (e.g., red blood cells, plasma and platelets). The term“apheresis” refers broadly to procedures in which blood is removed froma donor and separated into various components, the component(s) ofinterest being collected and retained and the other components beingreturned to the donor. The donor receives replacement fluids during thereinfusion process to help compensate for the volume and pressure losscaused by component removal. Apherersis systems are described in PCTpublication WO96/40857, hereby incorporated by reference.

Low Molecular Weight Compounds

A device of the present invention reduces the concentration of a lowmolecular weight compound in a composition containing cells. The term“low molecular weight compound” refers to an organic or biologicalmolecule having a molecular weight ranging from about 100 g/mol to about30,000 g/mol. Low molecular weight compounds include, withoutlimitation, the following compounds: small organic compounds such aspsoralens, acridines or dyes; quenchers, such as glutathione; plasticextractables, such as plasticizers; biological modifiers, such asactivated complement, that possess a molecular weight between about 100g/mol and about 30,000 g/mol; and, polyamine derivatives.

Small Organic Compounds

A diverse set of small organic compounds can be adsorbed by the deviceof the present invention. The molecules can be cyclic or acyclic. In oneembodiment the compounds are preferably, cyclic compounds such aspsoralens, acridines or dyes. In another embodiment the compounds arethiols.

Nonlimiting examples of cyclic compounds include actinomycins,anthracyclinones, mitomyacin, anthramycin, and organic dyes andphotoreactive compounds such as benzodipyrones, fluorenes, fluorenones,furocoumarins, porphyrins, protoporphyrins, purpurins, phthalocyanines,hypericin, Monostral Fast Blue, Norphillin A, phenanthridines,phenazathionium salts, phenazines, phenothiazines, phenylazides,quinolines and thiaxanthenones. Preferably the compounds arefurocoumarins or organic dyes. More preferably the compounds arefurocoumarins.

Nonlimiting examples of furocoumarins, include psoralens and psoralenderivatives. Specifically contemplated are4′-aminomethyl-4,5′,8-trimethylpsoralen, 8-methoxypsoralen, halogenatedpsoralens, isopsoralens and psoralens linked to quaternary amines,sugars, or other nucleic acid binding groups. Also contemplated are thefollowing psoralens: 5′-bromomethyl-4,4′,8-trimethylpsoralen,4′-bromomethyl-4,5′,8-trimethylpsoralen,4′-(4-amino-2-aza)butyl-4,5′,8-trimethylpsoralen,4′-(4-amino-2-oxa)butyl-4,5′,8-trimethylpsoralen,4′-(2-aminoethyl)-4,5′,8-trimethylpsoralen,4′-(5-amino-2-oxa)pentyl-4,5′,8-trimethylpsoralen,4′-(5-amino-2-aza)pentyl-4,5′,8-trimethylpsoralen,4′-(6-amino-2-aza)hexyl-4,5′,8-trimethylpsoralen,4′-(7-amino-2,5-oxa)heptyl-4,5′,8-trimethylpsoralen,4′-(12-amino-8-aza-2,5-dioxa)dodecyl-4,5′,8-trimethylpsoralen,4′-(13-amino-2-aza-6,11-dioxa)tridecyl-4,5′,8-trimethylpsoralen,4′-(7-amino-2-aza)heptyl-4,5′,8-trimethylpsoralen,4′-(7-amino-2-aza-5-oxa)heptyl-4,5′,8-trimethylpsoralen,4′-(9-amino-2,6-diaza)nonyl-4,5′,8-trimethylpsoralen,4′-(8-amino-5-aza-2-oxa)octyl-4,5′,8-trimethylpsoralen,4′-(9-amino-5-aza-2-oxa)nonyl-4,5′,8-trimethylpsoralen,4′-(14-amino-2,6,11-triaza)tetradecyl-4,5′,8-trimethylpsoralen,5′-(4-amino-2-aza)butyl-4,4′,8-trimethylpsoralen,5′-(6-amino-2-aza)hexyl-4,4′,8-trimethylpsoralen and5′-(4-amino-2-oxa)butyl-4,4′,8-trimethylpsoralen. Preferably, thepsoralen is 4′-(4-amino-2-oxa)butyl-4,5′,8-trimethylpsoralen.

Acridines

Nonlimiting examples of acridines include acridine orange, acriflavine,quinacrine, N1,N1-bis(2-hydroxyethyl)-N4-(6-chloro-2-methoxy-9-acridinyl)-1,4-pentanediamine,9-(3-hydroxypropyl)aminoacridine, N-(9-acridinyl)glycine,S-(9-acridinyl)-glutathione. In a preferred embodiment the acridine isN-(9-acridinyl)-β-alanine, alternatively, named5-[(β-carboxyethyl)amino]acridine.

Dyes

Nonlimiting examples of dyes include phenothiazines such as methyleneblue, neutral red, toluidine blue, crystal violet and azure A,phenothiazones such as methylene violet Bernthsen, phthalocyanines suchas aluminum 1,8,15,22-tetraphenoxy-29H,31H-phthalocyanine chloride andsilica analogues, and hypericin. Preferably, the dye is methylene blueor toluidine blue. More preferably, the dye is methylene blue.

The term “thiazine dyes” includes dyes that contain a sulfur atom in oneor more rings. The most common thiazine dye is methylene blue[3,7-Bis(dimethylamino)-phenothiazin-5-ium chloride). Other thiazinedyes include, but are not limited to, azure A, azure C and thionine, asdescribed e.g. in U.S. Pat. No. 5,571,666 to Schinazi.

The term “xanthene dyes” refers to dyes that are derivatives of thecompound xanthene. The xanthene dyes may be placed into one of threemajor categories: i) fluorenes or amino xanthenes, ii) the rhodols oraminohydroxyxanthenes, and iii) the fluorones or hydroxyxantheses.Examples of xanthene dyes contemplated for use with the presentinvention include rose bengal and eosin Y; these dyes may becommercially obtained from a number of sources (e.g., Sigma ChemicalCo., St. Louis, Mich.), and as described e.g. in U.S. Pat. No. 5,571,666to Schinazi, hereby incorporated by reference.

Quenchers

The concentration of a variety of compounds may be reduced. Otherexemplary compounds include quenching compounds. Methods for quenchingundesired side reactions of pathogen inactivating compounds that includea functional group which is, or which is capable of forming, anelectrophilic group, are described in the co-owned U.S. PatentApplication, “Methods for Quenching Pathogen Inactivators in BiologicalSystems”, 60/070,597, filed Jan. 6, 1998, the disclosure of which isincorporated herein. In this method, a material, such as a bloodproduct, is treated with the pathogen inactivating compound and aquencher, wherein the quencher comprises a nucleophilic functional groupthat is capable of covalently reacting with the electrophilic group. Inone embodiment, the pathogen inactivating compound includes a nucleicacid binding ligand and a functional group, such as a mustard group,which is capable of reacting in situ to form the electrophilic group.Examples of quenchers include, but are not limited to, compoundsincluding nucleophilic groups. Exemplary nucleophilic groups includethiol, thioacid, dithoic acid, thiocarbamate, dithiocarbamate, amine,phosphate, and thiophosphate groups. The quencher may be, or contain, anitrogen heterocycle such as pyridine. The quencher can be a phosphatecontaining compound such as glucose-6-phosphate. The quencher also canbe a thiol containing compound, including, but not limited to,glutathione, cysteine, N-acetylcysteine, mercaptoethanol, dimercaprol,mercaptan, mercaptoethanesulfonic acid and salts thereof, e.g., MESNA,homocysteine, aminoethane thiol, dimethylaminoethane thiol,dithiothreitol, and other thiol containing compounds. Exemplary aromaticthiol compounds include 2-mercaptobenzimidazolesulfonic acid,2-mercapto-nicotinic acid, napthalenethiol, quinoline thiol,4-nitro-thiophenol, and thiophenol. Other quenchers includenitrobenzylpyridine and inorganic nucleophiles such as selenide salts ororganoselenides, thiosulfate, sulfite, sulfide, thiophosphate,pyrophosphate, hydrosulfide, and dithionitrite. The quencher can be apeptide compound containing a nucleophilic group. For example, thequencher may be a cysteine containing compound, for example, adipeptide, such as GlyCys, or a tripeptide, such as glutathione.

Compounds that may be removed by the device of the present invention mayinclude thiols such as methyl thioglycolate, thiolactic acid,thiophenol, 2-mercaptopyridine, 3-mercapto-2-butanol,2-mercaptobenzothiazole, thiosalicylic acid and thioctic acid.

Plastic Extractables

The concentration of a group of low molecular weight compounds that areextractables from plastic storage containers and tubing used to handlebiological compositions may also be reduced in a biological compositionusing a device of the present invention. Examples of extractablesinclude, but are not limited to, plasticizers, residual monomer, lowmolecular weight oligomers, antioxidants and lubricants. See, e.g., R.Carmen, Transfusion Medicine Reviews 7(1):1-10 (1993). The sterilizationof plastic components by steam, gamma irradiation or electron beam canproduce oxidative reactions and/or polymer scission, resulting in theformation of additional extractable species.

Plasticizers are commonly used to enhance properties of plastics such asprocessability and gas permeability. The most common plasticizer foundin blood storage containers is di(2-ethylhexyl) phthalate (DEHP), whichis used in PVC formulations. DEHP has been identified as a potentialcarcinogen. Alternative plasticizers have been developed, including,without limitation, the following compounds: tri(2-ethylhexyl)trimellitate (TEHTM), acetyl-tri-n-hexyl citrate (ATHC),butyryl-tri-n-hexyl-citrate (BTHC), and di-n-decyl phthalate.

A device of the present invention may be used to reduce or control theconcentration of plastic extractables in a biological composition in avariety of settings. Such settings include, but are not limited to, thefollowing: blood treatment; blood storage; and, extracorporealapplications such as hemodialysis and extracorporeal membraneoxygenation.

Biological Response Modifiers (BRMs)

The concentration of a group of low molecular weight compounds broadlyreferred to as biological response modifiers (BRMs) may also be reducedor controlled in a biological composition using a device of the presentinvention. BRMs are defined as “a wide spectrum of molecules that alterthe immune response.” Illustrated Dictionary of Immunology, J. M. Cruseand R. E. Lewis. General groups of BRMs include, without limitation, thefollowing types of compounds: small molecules such as histamine andserotonin; lipids such as thromboxanes, prostaglandins, leukotrienes andarachidonic acid; small peptides such as bradykinin; larger polypeptidesthat contain further groups, including activated complement fragments(C3a, C5a); cytokines such as IL-1, IL-6 and IL-8; and chemokines suchas RANTES and MIP.

The accumulation of BRMs in a blood product during storage can adverselyaffect the desired biological activity of a biological composition.Complement activation, for example, has been demonstrated to occurduring storage of platelets under standard blood bank conditions.Complement activation has been associated with a loss of plateletfunction and viability termed “platelet storage lesion.” See, e.g., V.D. Mietic and O. Popovic, Transfusion 33(2):150-154 (1993). Theaccumulation of BRMs in a stored blood products can also, for example,adversely affect a patient that receives the blood product: theaccumulation of BRMs in platelet concentrates during storage has beenassociated with non-hemolytic febrile transfusion reactions in patientsreceiving platelets. See, e.g., N. M. Heddle, Current Opinions inHematology 2(6):478-483 (1995).

Polyamine Derivatives

The concentration of a group of low molecular weight compounds known aspolyamine derivatives may also, for example, be reduced in a biologicalcomposition using a device of the present invention. Polyaminederivatives are compounds that contain multiple nitrogen atoms in acarbon backbone.

Polyethylene Glycols

Other exemplary compounds include activated polyethylene glycols (aPEG),which may be used for the modification of the surface of cells ormaterials in order to provide immunomasking properties or pacificationtoward protein binding, respectively. The device may be used for thereduction of either the excess activated polyethylene glycol or theunreactive derivative of the PEG resulting from the reaction of theactivated PEG with water or small nucleophiles such as phosphate,phosphate esters or thiols, such as glutathione. Other compounds thatmay be removed include impurities in the activated PEG preparation,which may affect the function of the blood products or make themunsuitable for transfusion (eg. toxic compounds). Finally, smallmolecules (leaving groups) such as N-Hydroxy succinimide which arereleased during the reaction of the aPEG with cell surface nucleophilesmay also be reduced.

Examples of compounds that may be removed by the device of the presentinvention include linear or branched polyethylene glycols attached toactivating moeities which may include cyanuryl chloride, succinimidylesters, oxycarbonyl imidazole derivatives, nitrophenyl carbonatederivatives, glycidyl ether derivatives, and aldehydes.

It is to be understood that the invention is not to be limited to theexact details of operation or exact compounds, composition, methods, orprocedures shown and described, as modifications and equivalents will beapparent to one skilled in the art. From the above, it should be clearthat the methods and devices can be incorporated with apheresis systemsand other devices and procedures currently used to process bloodproducts for transfusion.

EXAMPLES

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); g (grams); mg (milligrams); μg (micrograms); Kg(kilograms); L (liters); mL (milliliters); μL (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); min.(minutes); s and sec. (seconds); J (Joules, also watt second); ZC(degrees Centigrade); TLC (Thin Layer Chromatography); HPLC (highpressure liquid chromatography); pHEMA and p(HEMA) (poly[2-hydroxyethylmethacrylate]); PC(s) (platelet concentrate(s)); PT (prothrombin time);aPTT (activated partial thromboplastin time); TT (thrombin time); HSR(hypotonic shock response); FDA (United States Food and DrugAdministration); GMP (good manufacturing practices); DMF (DrugMasterfiles); SPE (Solid Phase Extraction); Aldrich (Milwaukee, Wis.);Asahi (Asahi Medical Co., Ltd., Tokyo, Japan); Baker (J. T. Baker, Inc.,Phillipsburg, N.J.); Barnstead (Barnstead/Thermolyne Corp., Dubuque,Iowa); Becton Dickinson (Becton Dickinson Microbiology Systems;Cockeysville, Md.); Bio-Rad (Bio-Rad Laboratories, Hercules, Calif.);Cerus (Cerus Corporation; Concord, Calif.); Chrono-Log (Chrono-LogCorp.; Havertown, Pa.); Ciba-Corning (Ciba-Corning Diagnostics Corp.;Oberlin, Ohio); Consolidated Plastics (Consolidated Plastics Co.,Twinsburg, Ohio); Dow (Dow Chemical Co.; Midland, Mich.); Eppendorf(Eppendorf North America Inc., Madison, Wis.); Gelman (Gelman Sciences,Ann Arbor, Mich.); Grace Davison (W.R. Grace & Co., Baltimore, Md.);Helmer (Helmer Labs, Noblesville, Ind.); Hoechst Celanese (HoechstCelanese Corp., Charlotte, N.C.); International Processing Corp.(Winchester, Ky.); Millipore (Milford, Mass.); NIS (Nicolet, a ThermoSpectra Co., San Diego, Calif.); Poretics (Livermore, Calif.); Purolite(Bala Cynwyd, Pa.); Rohm and Haas (Chauny, France); Quidel (San Diego,Calif.); Saati (Stamford, Conn.); Scientific Polymer Products (Ontario,N.Y.); Sigma (Sigma Chemical Company, St. Louis, Mo.); Spectrum(Spectrum Chemical Mfg. Corp., Gardenia, Calif.); Sterigenics (Corona,Calif.); Tetko, Inc. (Depew, N.Y.); TosoHaas (TosoHass, Montgomeryville,Pa.); Wallac (Wallac Inc., Gaithersburg, Md.); West Vaco (Luke, W.Va.);YMC(YMC Inc., Wilmington, N.C.); DVB (divinyl benzene); LAL (LimulusAmoebocyte Lystate); USP (United States Pharmacopeia); EAA(ethyl-acetoacetate); EtOH (ethanol); HOAc (acetic acid); W (watts); mW(milliwatts); NMR (Nuclear Magnetic Resonance; spectra obtained at roomtemperature on a Varian Gemini 200 MHz Fourier Transform Spectrometer);ft³/min (cubic feet per minute); m.p. (melting point); g/min and gpm(gallons per minute); UV (ultraviolet light); THF (tetrahydrofuran);DMEM (Dulbecco's Modified Eagles Medium); FBS (fetal bovine serum); LB(Luria Broth); EDTA (ethelene diamine tetracidic acid); PhorbolMyristate Acetate (PMA); phosphate buffered saline (PBS); AAMI(Association for the Advancement of Medical Instruments); ISO(International Standards Organization); EU (endotoxin units); LVI (largevolume injectables); GC (gas chromatography); M (mega-); kGy (1000Gray=0.1 MRad); MΩ (Mohm); PAS III (platelet additive solution III);dH₂O (distilled water); IAD (immobilization adsorption device); SCD(sterile connection [connect] device).

One of the examples below refers to HEPES buffer. This buffer contains8.0 g of 137 mM NaCl, 0.2 g of 2.7 mM KCl, 0.203 g of 1 mM MgCl₂(6H₂0),1.0 g of 5.6 mM glucose, 1.0 g of 1 mg/ml Bovine Serum Albumin (BSA)(available from Sigma, St. Louis, Mo.), and 4.8 g of 20 mM HEPES(available from Sigma, St. Louis, Mo.).

Example 1 Fiberized Resin with Amberlite® XAD-16

This example compares both the kinetics of removal of aminopsoralensfrom platelets and platelet function and morphology utilizing fiberizedresin and devices containing non-immobilized adsorbent beads. Morespecifically, fiberized resin comprising immobilized Amberlite® XAD-16was compared with devices containing free (i.e., not immobilized)Amberlite® XAD-16 HP and Dowex® XUS-43493.

Preparation of Fiberized Resin and Adsorbent Beads

Immobilized adsorbent media containing Amberlite® XAD-16 in a cleanedand hydrated state (Rohm and Haas) was obtained from AQF. The fibers ofHoechst Celanese's fiber network consisted of a polyethyleneterephthalate core and a nylon sheath, the sheath having a lower meltingtemperature than the core. The fiberized resin was prepared by firstevenly distributing the adsorbent beads in the fiber network. Next, thefiber network was rapidly heated causing the polymer sheath of thefibers to melt and bond to the adsorbent beads and other fibers, forminga cross-linked fiber network. The fiberized resin formed contained theAmberlite® XAD-16 at a loading of 130 g/m² (i.e., each square meter offiber contained 130 g of adsorbent beads).

The fiberized resin was cut into squares (14 cm×14 cm), and theresulting sections contained approximately 2.5 g of dry Amberlite®XAD-16. The Amberlite® XAD-16 beads were then pre-wet by soaking thefiberized resin in 30% ethanol for approximately 10 minutes. Theresidual ethanol was then removed by rinsing twice in saline for 10minutes. Alternative methods of wetting the Amberlite® XAD-16 and otheradsorbents are also effective and are contemplated by the presentinvention. It should be noted that fiberized resin containing othertypes of beads (e.g., bridged or hypercrosslinked resins like Dowex®XUS-43493) do not require a wetting step for effective psoralen removal.

Amberlite® XAD-16 HP (High Purity) beads were also obtained directlyfrom Rohm and Haas in a cleaned and hydrated state. No pre-wetting wasrequired for the loose (i.e., not immobilized) Amberlite® XAD-16 HPbeads prior to incorporation into a mesh pouch; however, the mass ofadsorbent was corrected to account for the water content of the beads(2.5 g dry=6.8 g with 62.8% moisture). The Dowex® XUS-43493 beads wereobtained from Dow, and the dry beads did not require wetting nor did themass of the beads require correction for water. Polyester mesh pouches(7 cm×7 cm square; 30 μm openings) were then filled with 2.5 g (dryweight) of either the loose Amberlite® XAD-16 HP or Dowex® XUS-43493beads.

The fiberized resin and adsorbent-containing pouches were sterilized byautoclaving on “wet” cycle for 45 minutes at 121° C. Thereafter, thefiberized resin and the adsorbent-containing pouches were inserted intoseparate, sterile, 1-liter PL 2410 Plastic containers (Baxter).Following insertion, the PL 2410 Plastic containers were heat sealed ina laminar flow hood, using sterile scissors, hemostats, and an impulsesealer.

Contacting Fiberized Resin and Adsorbent Beads with Psoralen-ContainingPlatelet Concentrate (PC)

Pools of platelet concentrate were prepared by combining 2-3 units ofsingle donor apheresis platelets in 35% autologous plasma/65% PlateletAdditive Solution (i.e., synthetic media). To this solution was addedthe aminopsoralen 4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen(S-59) in an amount to achieve a final concentration of 150 μM4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen. The resulting PCsolution was divided into 300 mL units, and the units were then placedin PL 2410 Plastic containers (Baxter) and illuminated with 3 J/cm² ofUVA. Following illumination, the treated PCs were transferred into thePL 2410 Plastic containers containing either fiberized resin withimmobilized Amberlite® XAD-16, loose Amberlite® XAD-16 HP or looseDowex® XUS-43493, or into an empty PL 2410 Plastic container as acontrol. The PL 2410 Plastic containers (Baxter) were then placed on aHelmer platelet incubator at 22° C. and agitated at approximately 70cycles/minute.

Samples of each PC were removed at 1-hour intervals during the first 8hours of storage for analysis by HPLC of residual4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen. Each sample of PC wasdiluted 5-fold with sample diluent (final concentration=35% methanol, 25mM KH₂PO₄, pH=3.5) containing trimethylpsoralen (TMP) as the internalstandard. Proteins and other macromolecules were precipitated byincubating the samples at 4° C. for 30 minutes. The samples were thencentrifuged and the supernatant was filtered (0.2 μmeter) and analyzedon a C-18 reversed phase column (YMC ODS-AM 4.6 mm×250 mm) by running alinear gradient from 65% solvent A (25 mM KH₂PO₄, pH=3.5), 35% B(methanol) to 80% B in 20 minutes.

Platelet yield during a 5-day storage period with the fiberized resin orone of the loose beads was monitored daily by counting platelets on aBaker System 9118 CP (Baker Instrument Co.; Allentown, Pa.). Blood gasesand pH were evaluated using a Ciba-Corning 238 pH/Blood Gas Analyzer. Invitro platelet function following 5 days of contact with the fiberizedresin or the device containing free adsorbents was evaluated usingassays for morphology, shape change, hypotonic shock response,aggregation, and GMP-140 (p-selectin) expression. Shape change,aggregation, and hypotonic shock response were evaluated using aLumi-Aggregometer (Chrono-Log), while GMP-140 was determined by flowcytometry using a Becton-Dickinson FACScan Fluorescence Analyzer (BectonDickinson).

Psoralen Removal and Platelet Yield and Function

FIG. 5 compares the adsorption kinetics for removal of4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen from platelets in 35%plasma/65% synthetic media (PAS III) with XUS-43493, XAD-16 HP, andfiberized resin containing XAD-16. Specifically, the data indicated bythe circles connected by the solid line represents the device containingthe non-immobilized adsorbent XUS-43493 (2.5 g beads; <5% moisture); thedata indicated by the triangles connected by the dashed line representsthe device containing non-immobilized XAD-16 HP (6.8 g beads; 62.8%moisture); and the data indicated by the squares connected by the dashedline represents the fiberized resin (Hoechst fibers with XAD-16 beadswet in 30% ethanol; 14 cm×14 cm). As the data in FIG. 5 indicate, thekinetics of 4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen adsorptionare very comparable for both the device containing non-immobilizedadsorbents and device containing the fiberized resin. Thus, thefiberization process does not appear to have a significant impact on theremoval kinetics.

In addition, platelet yield and function of the fiberized resin comparedto the loose beads were studied. Specifically, the experiments of thisstudy used i) 6.8 g XAD-16 HP (62.8% moisture); ii) 14 cm×14 cmfiberized XAD-16 (130 g resin/cm²) wet in 30% ethanol; and iii) 2.5 gXUS-43493 (<5% moisture). Duplicate platelet units were prepared for theXAD-16 HP and the fiberized resin samples, but only a single plateletunit was prepared for the XUS-43493 sample. The results are set forth inTable 1.

As indicated in Table 1, the day-5 pH and pO₂ values were slightlyelevated relative to the day 5 control for samples containingnon-immobilized beads (XAD-16 HP and XUS-43493). The experiment with thefiberized resin had pH and pO₂ values which were more comparable to thecontrol. Platelet counts indicated a 9-22% platelet loss following 5days of contact in the control with the fiberized media and the devicecontaining non-immobilized adsorbents. As set forth in Table 1, thefiberized resin gave better yields (9% loss on day 5) and performedbetter in all in vitro assays when compared to device containingnon-immobilized XAD-16 or XUS-43493 adsorbent particles.

TABLE 1 Platelet Hypotonic Count Shape shock GMP-140 Sample pH pO₂ pCO₂(10¹¹/300 mL) change Aggreg response Morph (%) Control Day 0 6.98 56 314.40 ± 0.18 Control Day 5 6.89 80 27 4.21 ± 0.16 0.58 ± 0.03 83 ± 3 0.24± 0.02 297 60.6 XAD-16 HP 6.98 110 22 3.65 ± 0.09 0.38 ± 0.09 70 ± 60.29 ± 0.09 278 53.8 (−13.3%) IAD XAD-16 6.90 72 28 3.82 ± 0.15 0.56 ±0.12 80 ± 4 0.27 ± 0.04 292 55.8  (−9.3%) XUS-43493 6.98 115 20 3.27 ±0.05 0.37 ± 0.28 51 ± 2 0.64 ± 0.10 271 67.0 (−22.3%)

Though an understanding of the mechanism underlying the higher pH andpO₂ values observed for the device containing non-immobilized XUS-43493and XAD-16 HP is not required in order to practice the presentinvention, the higher values are believed to be caused by a slightdecrease in the metabolism of the platelets in the presence of thedevice containing non-immobilized adsorbent. By comparison, thefiberized media consistently gave day-5 pH and pO₂ values which weremore comparable to the control than the XUS-43493 or XAD-16 beads.

The day-5 platelet yields were also better for the fiberized mediarelative to the XUS-43493 and XAD-16 HP adsorbent beads. The 22-28% losswhich was observed for the XUS-43493 media was observed on severaloccasions. However, it should be noted that the current preferredembodiment for a device containing non-immobilized adsorbent withXUS-43493 involves transfer of the platelets from this device after 8hours of exposure; this procedure results in <5% loss in platelets.

The data presented in Table 1 indicates that the fiberized media gives ahigher platelet yield relative to devices containing non-immobilizedadsorbents. Surprisingly, the fiberized media also results in betterday-5 platelet function as indicated by pH/pO₂, shape change,aggregation, morphology and GMP-140. While an understanding of therationale for the enhanced performance of the fiberized media is notrequired to practice the present invention, several hypotheses can beproposed. First, the fibers which are attached to the surface of theadsorbent beads may hinder interaction between platelets and the surfaceof the beads. Second, immobilizing the beads may prevent the beads frominteracting and eliminate mechanical effects that are detrimental toplatelets. Third, immobilizing the beads may enhance fluid shear at thebead surface, thereby decreasing interaction between platelets and thesurface of the beads; by comparison, non-immobilized beads are free toflow with the fluid resulting in low flow of fluid relative to thesurface of the bead.

Platelet Loss

When reviewing the above data, it appears that there is some variabilityin platelet loss from one study to the next. However, the platelet lossexpressed as a percentage of the day-5 control count is smaller forstudies where the initial platelet count is higher. A study wasperformed in order to confirm whether the number of platelets that arelost is constant for a given area of material available for plateletadhesion. For this study, two platelet units were pooled and the poolwas divided into two samples. One sample was diluted in half with 35%autologous plasma/65% synthetic media (PAS) so that the platelet countwas half of the other unit. The platelet mixtures were treated with4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen+UVA and were contactedwith a device containing non-immobilized adsorbent (2.5 g XUS-43493) for5 days under the previously discussed standard platelet storageconditions.

The total number of platelets that was lost was virtually identical forthe two units, while the losses calculated as a percent differedgreatly. Thus, the results indicate that total platelet loss appears tobe essentially constant after a period of time; that is, while thepercentage of platelet loss varies with the initial platelet count, thetotal number of platelets lost will be approximately constant whenequilibrium is reached. Based on the results set forth in this example,the fiberized resin does not have a negative effect on in vitro plateletfunction.

Example 2 Fiberized Resin with Activated Charcoal

This example compares the kinetics of removal of4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen from platelets andplatelet function and morphology for fiberized resin comprisingAmberlite® XAD-16 and for fiberized resin comprising immobilizedactivated charcoal.

Preparation of Fiberized Resin

Hoechst Celanese prepared fiberized resin containing Amberlite® XAD-16HP (Rohm and Haas). The fiberized resin containing the Amberlite® XAD-16was prepared as described in the preceding example, including the 30%ethanol wetting step. Hoechst Celanese also prepared fiberized resincontaining immobilized activated charcoal (WestVaco) at a loading of 375g/m² (AQF-375-B) and 500 g/m² (AQF-500-B). #In a preferred embodiment,the adsorbent particles is a synthetic activated carbon, including forexample, Ambersorb and A-Supra. Synthetic activated carbons arepreferred due to their ability to eliminate the amount of particulatematerial shed from the immobilized adsorption medium. this fiberizedresin was prepared in a method analogous to that for the fiberized resincontaining Amberlite® XAD-16. The composition of the fibers for eachfiberized resin was the same.

The fiberized resin was cut into squares (14 cm×14 cm); the resultingsections contained approximately 2.5 g of dry Amberlite® XAD-16. Next,the fiberized resin was sterilized by autoclaving on “wet” cycle for 45minutes at 121° C. Thereafter, the fiberized resin were inserted intoseparate sterile, 1-liter PL 2410 Plastic containers (Baxter), and thecontainers were heat sealed in a laminar flow hood, using sterilescissors, hemostats, and an impulse sealer.

Contacting Fiberized Resin with Psoralen-Containing PC

Pools of platelet concentrate were prepared by combining 2-3 units ofsingle donor apheresis platelets in 35% autologous plasma/65% syntheticmedia (PAS III). 4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen wasadded in an amount to achieve a final concentration of 150 μM4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen. The resulting PCsolution was divided into 300 mL units, and the units were placed in PL2410 Plastic containers (Baxter) and illuminated with 3 J/cm² of UVA.Following illumination, the treated PCs were transferred into the PL2410 Plastic containers containing fiberized resin with either XAD-16,AQF-375-B, AQF-500-B or into an empty PL 2410 Plastic container as acontrol. The PL 2410 Plastic containers were then placed on a Helmerplatelet incubator at 22° C. and agitated at approximately 70cycles/minute.

As performed in the preceding example, samples of each PC were removedat 1-hour intervals during the first 8 hours of storage for analysis ofresidual 4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen by HPLC. Eachsample of PC was diluted 5-fold with sample diluent (finalconcentration=35% methanol, 25 mM KH₂PO₄, pH=3.5) containingtrimethylpsoralen (TMP) as the internal standard. Proteins and othermacromolecules were precipitated by incubating the samples at 4° C. for30 minutes. The samples were then centrifuged and the supernatant wasfiltered (0.2 μmeter) and analyzed on a C-18 reversed phase column (YMCODS-AM 4.6 mm×250 mm) by running a linear gradient from 65% solvent A(25 mM KH₂PO₄, pH=3.5), 35% B (methanol) to 80% in 20 minutes.

Platelet yield during a 5-day storage period with the fiberized resin orthe device containing non-immobilized adsorbent was monitored daily bycounting platelets on a Baker System 9118 CP. Blood gases and pH wereevaluated using a Ciba-Corning 238 pH/Blood Gas Analyzer. In vitroplatelet function following 5 days of contact with the fiberized resinor the device containing non-immobilized adsorbent was evaluated usingassays for morphology, shape change, hypotonic shock response,aggregation, and GMP-140 (p-selectin) expression. Shape change,aggregation, and hypotonic shock response were evaluated using aChrono-Log Lumi-Aggregometer, while GMP-140 was determined by flowcytometry using a Becton-Dickinson FACScan Fluorescence Analyzer.

Psoralen Removal and Platelet Yield and Function

FIG. 6 compares the adsorption kinetics for removal of4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen from platelets in 35%plasma/65% synthetic media (PAS III) with fiberized resin containingXAD-16 and fiberized resin with the two different loadings of activatedcharcoal. Specifically, the data indicated by the circles represents4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen removal with fiberizedXAD-16 beads; the data indicated by the squares represents removal withfiberized AQF-500-B; and the data indicated by the triangles representsremoval with fiberized AQF-375-B. As the data in FIG. 6 indicate, thekinetics of 4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen adsorptionare very comparable for the different fiberized resin, and the fiberizedresin all showed very good kinetics of removal (≦0.5 μM residual4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen after 4 hours).

Platelet yield and in vitro platelet function for each of the fiberizedresin were also evaluated and the data are summarized in Table 2.

TABLE 2 Platelet Count Shape Sample pH pO₂ pCO₂ (10¹¹/300 mL) ChangeAggregation Control Day 0 7.07 59 23 3.34 ± 0.13 1.17 ± 0.07 90 ± 5Control Day 5 6.88 112 21 3.09 ± 0.22 0.52 ± 0.02 72 ± 8 Fiberized Resin6.93 117 20 2.55 ± 0.16 0.39 ± 0.13 69 ± 6 with XAD-16 (−17%)  FiberizedResin 7.07 100 20 2.83 ± 0.04 0.74 ± 0.05 61 ± 0 with AQF-500-B (−8%)Fiberized Resin 6.99 107 20 2.82 ± 0.16 0.46 ± 0.06 65 ± 2 withAQF-375-B (−9%)

Referring to Table 2, the charcoal-based fiberized resin gave goodplatelet yields with losses of less than 10%; as in the studies of thepreceding example, the fiberized resin containing XAD-16 had a slightlyhigher platelet loss (about 17%). Regarding pO₂, the day 5 values forthe charcoal fiberized resin are comparable to the control. Though anunderstanding of why the charcoal fiberized resin had slightly elevatedpH values is not required to practice the present invention, it may bean artifact caused by residual extractables (e.g., phosphate) from theactivation process; the rapid rise in pH (pH=7.3-7.4) observed after 8hours of storage of the PC with the charcoal-based fiberized resinsupports that idea. The use of USP charcoals, which are associated withfewer extractables, may eliminate the observed initial rise in pH.

The charcoal-based fiberized resin provided good results in both theshape change and aggregation assays. Although the shape change resultfor the AQF-500-B fiberized resin is better than that of the control,the platelets associated with AQF-500-B performed slightly poorer in theaggregation assay.

Example 3 Effect of pHEMA Coating on Adsorbent Hemocompatibility

This example compares the kinetics of removal of4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen from platelets andplatelet function and morphology for both Dowex® XUS-43493 and fiberizedresin containing Amberlite® XAD-16 coated with pHEMA.

Preparation of pHEMA-Coated Adsorbent Beads and Fiberized Resin

Dowex® XUS-43493 (commercially known as Optipore® L493) containingapproximately 50% water by weight was obtained from Dow, and polymerizedHEMA with a viscosity average molecular weight of 300 kD was obtainedfrom Scientific Polymer Products. Prior to coating, the adsorbent beadswere dried to a water content of <5%. A stock solution of pHEMA wasprepared by dissolving the polymer in 95% denatured ethanol/5% water toachieve a pHEMA concentration of 50 mg/ml.

The coating process was performed by International Processing Corp. in a9-inch Wurster fluidized bed coater with a charge of approximately 4 kg(dry) of adsorbent. The coating process involved a pHEMA flow rate of60-70 g/min, an inlet temperature of 50° C., and an air flow rate ofapproximately 200 ft³/min. Samples (50 g) of coated adsorbent wereremoved during the coating process so that coating levels ranging from3-18% (w/w) pHEMA were obtained; adsorbent beads coated with 3.7%, 7.3%,and 10.9% pHEMA (w/w) were used in the studies described below.

A device containing non-immobilized dry (uncoated) Dowex® XUS-43493 (2.5g) and pHEMA-coated Dowex® XUS-43493 (3.0 g or 5.0 g) were prepared byplacing the desired mass of adsorbent into a square 30 μm polyester meshpouch (7 cm×7 cm). The adsorbent-filled pouches were inserted intoseparate sterile, 1-liter PL 2410 Plastic containers (Baxter) and heatsealed with an impulse sealer. Thereafter, the adsorbent-filled pouchescontaining PL-2410 Plastic containers were sterilized by either E-beam(NIS) or gamma irradiation (SteriGenics) to 2.5 MRad; as previouslyalluded to, E-beam sterilization is generally preferred.

Hoechst Celanese prepared fiberized resin containing Amberlite® XAD-16according to the method described in Example 1. The fiberized resin wascut into squares (14 cm×14 cm); the resulting sections containedapproximately 2.5 g of dry Amberlite® XAD-16. The Amberlite® XAD-16 ofthe fiberized resin was simultaneously wet and coated with pHEMA bysoaking in a solution containing 50 mg/mL pHEMA in 95% ethanol/5%distilled water. Residual ethanol was removed by rinsing twice in salinefor 10 minutes. This procedure resulted in a coating of approximately 6%(w/w) pHEMA. The fiberized resin was then sterilized by autoclaving on“wet” cycle for 45 minutes at 121° C. Thereafter, the fiberized resinwas inserted into separate sterile, 1-liter PL 2410 Plastic containers(Baxter) and heat sealed in a laminar flow hood, using sterile scissors,hemostats, and an impulse sealer.

Contacting pHEMA-Coated Adsorbent Beads and Fiberized Resin withPsoralen-Containing PC

Pools of platelet concentrate were prepared by combining units of singledonor apheresis platelets in 35% autologous plasma/65% synthetic media(PAS III). 4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen was addedin an amount to achieve a final concentration of 150 μM4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen. The resulting PCsolution was then divided into 300 mL units, and the units were placedin PL 2410 Plastic containers (Baxter) and illuminated with 3 J/cm² ofUVA. Following illumination, the treated PCs were transferred into thePL 2410 Plastic containers containing the devices as indicated in thefollowing result sections. Control samples without an adsorption devicewere also prepared. The PL 2410 Plastic containers were then placed on aHelmer platelet incubator at 22° C. and agitated at approximately 70cycles/minute.

Samples of each PC were removed at 1-hour intervals during the first 8hours of storage for analysis of residual4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen by HPLC. Each sampleof PC was diluted 5-fold with sample diluent (final concentration=35%methanol, 25 mM KH₂PO₄, pH=3.5) containing trimethylpsoralen (TMP) asthe internal standard. Proteins and other macromolecules wereprecipitated by incubating the samples at 4° C. for 30 minutes. Thesamples were then centrifuged and the supernatant was filtered (0.2μmeter) and analyzed on a C-18 reversed phase column (YMC ODS-AM 4.6mm×250 mm) by running a linear gradient from 65% solvent A (25 mMKH₂PO₄, pH=3.5), 35% B (methanol) to 80% in 20 minutes.

Platelet yield after a 5-day storage period with the fiberized resin orthe device containing non-immobilized adsorbent was determined bycounting platelets on a Baker System 9118 CP. Blood gases and pH wereevaluated using a Ciba-Corning 238 pH/Blood Gas Analyzer. In vitroplatelet function following 5 days of contact with the fiberized resinor the control device containing non-immobilized adsorbent was evaluatedusing assays for morphology, shape change, hypotonic shock response,aggregation, and GMP-140 (p-selectin) expression. Shape change,aggregation, and hypotonic shock response were evaluated using aChrono-Log Lumi-Aggregometer, while GMP-140 was determined by flowcytometry using a Becton-Dickinson FACScan Fluorescence Analyzer.

Effect of pHEMA Coating on Psoralen Removal

FIG. 7 compares the adsorption kinetics for removal of4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen from platelets in 35%plasma/65% synthetic media (PAS III) with pHEMA-coated and uncoatedDowex® XUS-43493 beads. Specifically,4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen removal with 3.0 gDowex® XUS-43493 coated with 3.7% (w/w) pHEMA is represented by thecircles, with 7.3% (w/w) pHEMA is represented by the triangles, and with10.9% (w/w) pHEMA is represented by the diamonds; the squares represent4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen removal with 2.5 g(dry) uncoated Dowex® XUS-43493. As the data in FIG. 7 indicate, thekinetics of 4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen adsorptiondecreased as the level of pHEMA coating was increased. While themechanism need not be understood in order to practice the invention,this decrease is believed to be due to an increase in resistance todiffusion of 4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen to theinterior of the adsorbent particles.

The adsorption kinetics of 4′-(4-amino-2-oxa)butyl-4,5′,8-trimethylpsoralen removal were also determined with 5.0 g Dowex® XUS-43493 coatedwith 3.7%, 7.3%, and 10.9% (w/w) pHEMA. The results (not shown)indicated that the removal kinetics for the beads coated with 10.9%pHEMA were comparable to that with the uncoated (control) beads.

Effect of pHEMA Coating on Platelet Yield

The effect of pHEMA on platelet yield was determined in two studiesusing different amounts of adsorbent (3.0 and 5.0 g) but the same levelsof pHEMA coating (3.7%, 7.3%, and 10.9% [w/w]) in each. Platelet yieldswere calculated relative to the platelet count on day 5 for treated PCwhich was not contacted with a device containing non-immobilizedadsorbents. As the results in Table 3 indicate, when 3.0 g of XUS-43493were tested, there was a nominal dose response on day 5 platelet yieldwith increasing pHEMA coating levels; those results suggest that a lowlevel of pHEMA coating may be most effective since it has a smallereffect on 4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen removalkinetics while still inhibiting platelet adhesion to the adsorbentsurface. In contrast to the nominal effect seen with 3.0 g of XUS-43493,a dose response was observed when 5.0 g were tested—increasing pHEMAcoating levels did increase the day 5 platelet yield. However, yieldswere still lower than those observed when 3.0 g of adsorbent beads wereused.

TABLE 3 Polymer Coating Adsorbent Platelet Yield, Coating Level (%) Mass(g) Day 5 (%)* None 0 2.5 73.1 pHEMA 3.7 3.0 91.4 pHEMA 7.3 3.0 87.7pHEMA 10.9 3.0 89.0 pHEMA 3.7 5.0 71.8 pHEMA 7.3 5.0 80.2 pHEMA 10.9 5.086.6

The results presented in Table 3 suggest that the use of a lower mass ofadsorbent (e.g., 2.5-3.0 g) along with a low level of pHEMA coating(e.g., 3.0% w/w) will provide the best platelet yield. As previouslyindicated, the optimum level of pHEMA coating is the minimum coating atwhich a protective effect on platelet yield and in vitro plateletfunction is observed.

Effect of pHEMA Coating and Sterilization on Platelet Function

It was previously indicated that methods of sterilization may have asubstantial effect on adsorbent function since the pHEMA coating can becrosslinked or cleaved by the radiation. In order to evaluate thiseffect, a study was performed with devices containing non-immobilizedpHEMA-coated (3.7% w/w) and uncoated XUS-43493 sterilized with either2.5 MRad E-beam or 2.5 MRad gamma irradiation. Each device contained 2.5g (dry) of non-immobilized coated or uncoated XUS-43493 housed in asquare 30 μm polyester mesh pouch (7 cm×7 cm); the control comprisedtreated PC stored in a PL 2410 Plastic container alone. The results aresummarized in Table 4.

TABLE 4 Platelet GM-140 Count Shape Activation Sample pH pO₂ (10¹¹/300L) Change Aggregation HSR Morphology (%) Control 7.03 67 4.94 ± 0.24 — —— — — Day 0 Control 6.87 84 4.58 ± 0.08 0.96 ± 0.05 76 ± 2 0.35 ± 0.03295 70.0 Day 5   (−0%) Uncoated 6.95 128 3.49 ± 0.08 0.38 ± 0.00 44 ± 00.47 ± 0.12 260 58.3 XUS-43493 (−23.8%)  3.7% 6.93 108 4.19 ± 0.09 0.59± 0.03 59 ± 5 0.32 ± 0.04 240 58.5 pHEMA (−8.5%) XUS-43493 Gamma 3.7%6.88 92 4.35 ± 0.11 0.71 ± 0.06 69 ± 9 0.40 ± 0.04 264 54.8 pHEMA(−5.0%) XUS-43493 E-beam

Referring to Table 4, the day-5 pH values appear to be very stablecompared to the control. The pO₂ value measured with the devicecontaining non-immobilized uncoated adsorbent is slightly elevated,suggesting a mild decrease in metabolism; coating with pHEMA appeared toreduce this effect, with the results for the E-beam sterilized devicecontaining non-immobilized adsorbent closer to the control than thosefor the gamma sterilized device.

Platelet yields were very good for both of the pHEMA-coated samples, theE-beam sterilized sample performing slightly better. Shape change andaggregation exhibited a pattern similar to that for yield, with thedevice containing non-immobilized uncoated adsorbent giving the lowestvalues and the pHEMA-coated/E-beam sterilized sample providing highervalues similar to the control. The samples treated with a devicecontaining non-immobilized adsorbents performed as well as or betterthan the control in the hypotonic shock response (HSR) assay. Samplesthat were treated with the devices containing non-immobilized adsorbentsgave lower morphology scores than the control, but showed lower levelsof activation as indicated by the GMP-140 assay.

Another hemocompatibility study was performed comparing a devicecontaining non-immobilized uncoated XUS-43493 (2.5 g beads; 30 μmpolyester mesh pouch; 7 cm×7 cm), uncoated fiberized resin (14 cm×14 cm)containing Amberlite® XAD-16, and fiberized resin containingpHEMA-coated Amberlite®XAD-16. The favorable effect of pHEMA on thefiberized resin is indicated by the results presented in Table 5

TABLE 5 Platelet GM-140 Count Shape Activation Sample pH pO₂ (10¹¹/300L) Change Aggregation HSR Morphology (%) Control 7.11 50 3.01 ± 0.10 — —— — — Day 0 Control 6.91 122 2.85 ± 0.16 0.60 ± 0.09 78 ± 4 0.31 ± 0.01302 72.5 Day 5   (−0%) Uncoated 7.00 151 2.11 ± 0.07 0.35 ± 0.03 55 ± 40.57 ± 0.03 267 68.7 XUS- (−26.0%) 43493 Uncoated 6.94 126 2.23 ± 0.130.52 ± 0.03 82 ± 4 0.45 ± 0.01 297 65.3 Fiberized (−21.8%) Resin withXAD-16 pHEMA- 6.95 120 2.44 ± 0.11 0.59 ± 0.01 86 ± 5 0.46 ± 0.02 30563.0 Fiberized (−14.4%) Resin with XAD-16

As indicated by the data regarding in vitro platelet function andplatelet yield in Table 5, coating with pHEMA brought the pO₂ values forthe fiberized resin closer to that observed for the control. ThepHEMA-coated fiberized resin also exhibited higher platelet yield thanthe uncoated fiberized resin. The results indicate that the pHEMA-coatedfiberized resin performed better than the uncoated XUS-43493 beads inall in vitro platelet function assays. Moreover, the uncoated fiberizedresin performed better than the uncoated XUS-43493 beads in most invitro platelet function assays.

Example 4 Effect of Glycerol and Polyethylene Glycol on AdsorbentCapacity

This example examines the effect of glycerol and polyethylene glycol asstabilizing agents on adsorbent capacity and kinetics of removal ofaminopsoralens from plasma. Free (i.e., not fiberized) Amberlite® XAD-16and Dowex® XUS-43493 adsorbent beads were used in the experiments ofthis example.

Methodology

Amberlite® XAD-16 HP (Rohm & Haas (Philadelphia, Pa.)) andDowex®XUS-43493 (Supelco, Bellefonte, Pa.) were dried to <5% water in a80° C. oven. Known masses of adsorbent were soaked in ethanol solutionscontaining 0-50% glycerol, 50% PEG-200 or 50% PEG-400 (glycerol,PEG-200, and PEG-400 from Sigma). Following a 15 minute incubationperiod at room temperature, the excess solvent was removed and thesamples were dried overnight in a 80° C. oven; drying the adsorbent attemperatures>120° C. was avoided since changes in adsorbent properties(e.g., pore melting) were previously observed at higher temperatures.After drying, adsorbent samples were weighed to determine the mass ofstabilizing agent per mass of adsorbent.

Several individual studies were performed. Control samples of “non-wet”adsorbent and “optimally wet” adsorbent were included in the studies asdescribed below. The non-wet samples of adsorbent were dried adsorbentwhich was not subjected to any pre-treatment, while the optimally wetsamples of adsorbent were prepared by wetting the adsorbent with 30%ethanol/70% dH₂O. The optimally-wet adsorbent was rinsed with dH₂O toremove residual ethanol. The adsorbent was prepared just prior to theadsorption study to assure that drying did not occur.

Each of the adsorption studies was performed using 100% human plasmacontaining 150 μM 4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralenspiked with ³H-4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen. Plasma(6.0 mL) was added to vials containing adsorbent treated with differentstabilizing agents. Masses of adsorbent were corrected for glycerol orPEG content to give 0.2 g of adsorbent. The vials were placed on arotator and agitated at room temperature. Plasma samples were removed atvarious times and levels of residual³H-4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen were determined.Samples (200 μL) were diluted in 5.0 mL of Optiphase HiSafe LiquidScintillation Cocktail (Wallac) and were counted on a Wallac 1409 LiquidScintillation Counter (Wallac).

Adsorption Capacities of Amberlite® XAD-16 and Dowex® XUS-43493 Treatedwith Glycerol

FIG. 8 compares the effect of pre-treatment with ethanol solutionscontaining various levels of glycerol on relative4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen adsorption capacity in100% plasma for Amberlite® XAD-16 and Dowex® XUS-43493. Adsorbentsamples were wet in the ethanol/glycerol solutions for 15 minutes priorto drying for 48 hours at 80° C. Single measurements of adsorptioncapacity were made after 4 hours of contact. Referring to FIG. 8,glycerol content shown on the x-axis is weight/volume percent ofglycerol in ethanol. Adsorption capacities shown on the y-axis arepercentages relative to the adsorption capacity of the optimally wetadsorbent sample. The adsorption capacity of XUS-43493 is represented bythe squares, while that of XAD-16 is represented by the circles.

As the data in FIG. 8 indicate, the capacity of XAD-16 increased fromabout 30% in the dry sample to over 90% in the sample wet in a 20%glycerol solution. These results indicate that very low levels ofglycerol are required for maintaining high adsorbent capacity afterdrying. Control samples that were wet in 50% ethanol/50% dH₂O (noglycerol) prior to drying demonstrated adsorption capacities which weresimilar to untreated samples that were dried. In contrast, the XUS-43493samples did not show any effect of glycerol on adsorption capacity;adsorption capacity approached 100% at all levels of glycerol. While notcritical to the practice of the present invention, this observationsupports the hypothesis that glycerol acts to prevent the adsorbentpores from collapsing during drying; because XUS-43493 has a highlycrosslinked structure, it is not subject to pore collapse upon drying.

Samples that were treated with glycerol appeared to be very stable todrying. No changes were observed in adsorption capacity for samples thatwere stored for 7 days in a laminar flow hood (data not shown).

In a preferred embodiment of the present invention, 2.5 g dry ofadsorbent are used for the removal of psoralen and psoralenphotoproducts from each unit of platelets. Soaking the adsorbent in 30%glycerol/70% ethanol, followed by drying, results in adsorbent whichcontains approximately 50% glycerol. A 5.0 g sample of adsorbent wouldtherefore contain 2.5 g dry adsorbent and 2.5 g of glycerol. Thus, atypical 300 mL unit of platelets would contain 0.8% glycerol, a levelthought to be acceptable for transfusion.

Adsorption Capacities of Amberlite® XAD-16 and Dowex® XUS-43493 Treatedwith Glycerol or PEG

Additional studies were performed with the low molecular weightpolyethylene glycols PEG-200 and PEG-400, low-toxicity agents that arenonvolatile and are soluble in ethanol and water. Samples of adsorbentwere treated for 15 minutes in 50% solutions of PEG-400, PEG-200 orglycerol in ethanol. FIG. 9 compares the effect of the stabilizingagents on 4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen adsorptioncapacities with dried adsorbent in 100% plasma for Amberlite® XAD-16(bottom) and Dowex® XUS-43493 (top); the samples that were not wet arelabeled “No Tx”. Adsorbent capacities are reported as percentagesrelative to the capacity of optimally wet adsorbent.

As indicated by the data in FIG. 9 and predictable based on the its“macronet” structure, the capacity of Dowex® XUS-43493 was not affectedby drying (“No Tx” sample). Conversely, the Amberlite® XAD-16 hadapproximately 35% of the maximum capacity when dried. Treating XAD-16with glycerol, PEG-200, and PEG-400 all improved the capacity of thedried adsorbent; the adsorbent capacities with each were all greaterthan 90%, with glycerol>PEG-200>PEG-400. Though an understanding of theprecise mechanism of action is not required to practice the presentinvention, differences in capacity between the glycerol and the two PEGsolutions may be caused by decreasing penetration of the stabilizingagent with increasing molecular weight. That is, during the 15 minuteapplication procedure, the glycerol (MW=92.1) may be able to penetratethe adsorbent pores more completely than either PEG-200 (MW=190-210) orPEG-400 (MW=380-420), which diffuse more slowly because of their largersize.

Adsorption Kinetics of Amberlite® XAD-16 Treated with Glycerol or PEG

A study was also performed to determine whether filling the pores of theadsorbent with glycerol or PEG results in reduced adsorption kinetics.FIG. 10 compares adsorption of 4′-(4-amino-2-oxa)butyl-4,5′,8-trimethylpsoralen over a 3-hour period from 100% plasma using Amberlite® XAD-16wet in several different solutions. Specifically, the data in FIG. 10represents XAD-16 i) wet prior to drying with a 50% solution of glycerol(open squares connected by solid lines), ii) wet prior to drying with a50% solution of PEG-400 (shaded circles connected with dashed lines),iii) pre-wet, i.e., just prior to initiating the study, with 50%ethanol/50% dH₂O (shaded triangles connected by dashes), and iv) notsubjected to any treatment (shaded squares connected by solid lines; “NoTx”). The data in FIG. 10 demonstrate that Amberlite® XAD-16 samplesthat were wet in 50% glycerol/50% ethanol or 50% PEG-400/50% ethanolsolutions had adsorption kinetics which were very close to the samplethat was optimally wet in ethanol (i.e., the sample pre-wet withethanol). The XAD-16 sample that was dried but not treated (No Tx)achieved only about 30% removal by 3 hours.

The data presented in this example indicate that treating Amberlite®XAD-16 with stabilizing agents in the form of solutions containing 50%ethanol and 50% glycerol, PEG-200, or PEG-400 can prevent loss ofadsorption capacity associated with drying. The results obtained withthese stabilizing agents suggest that low molecular weight wettingagents represent viable methods for enhancing adsorbent function.

Example 5 Removal of Methylene Blue from FFP

This example is directed at the ability of a variety of differentpolymeric adsorbent materials to remove methylene blue from fresh frozenplasma.

The experiments of this example evaluated “free” adsorbent resin (i.e.,not incorporated into device containing non-immobilized adsorbents) andfiberized resin. The free adsorbent resins tested were Amberlite® XAD-16HP (Rohm and Haas), MN-200 (Purolite), and Dowex® XUS-43493 (DowChemical Co.). The XAD-16 HP came in a hydrated state so that nopre-treatment (i.e., no wetting) was necessary, and the MN-200 was alsosupplied in a fully hydrated state; the XUS-43493 was dry.

Fiberized resin containing XAD-16 was prepared as generally described inExample 1. Briefly, a 2 cm×7 cm (i.e., 14 cm²) strip of fiberized resincontaining 130 g/m² XAD-16 was first wet in 70% ethanol and then rinsedexhaustively in distilled water.

A stock solution of methylene blue (10 mM) was prepared by dissolvingU.S.P. methylene blue (Spectrum) in distilled water. The stock solutionof methylene blue was added to a sample of 100% plasma to give a finalconcentration of 10 μM. Samples of the “free” adsorbent resin (i.e.,XAD-16 HP, MN-200, and XUS-43493) were weighed into 50 mL polypropylenetubes for adsorption studies. The water content of each adsorbent wasdetermined by measuring mass loss upon drying. The mass of eachadsorbent was corrected for water content so that the equivalent of 0.25g dry adsorbent was used for each.

A 30 mL sample of the 100% plasma containing 10 μM methylene blue wasadded to each vial. The vials were placed on a rotator at roomtemperature. Samples (200 μL) were removed from each vial at 15 minuteintervals and assayed for residual methylene blue by HPLC. Each sampleof plasma was diluted 5-fold with sample diluent (finalconcentration=35% methanol, 25 mM KH₂PO₄, pH=3.5). Proteins and othermacromolecules were precipitated by incubating the samples at 4° C. for30 minutes. Samples were centrifuged and the supernatant was filtered(0.2 μm) and analyzed on a C-18 reversed phase column (YMC ODS-AM, 4.6mm×250 mm) by running a linear gradient from 65% solvent A (25 mMKH₂PO₄, pH=3.5), 35% B (Methanol) to 80% B in 20 minutes. The limit ofdetection for the HPLC assay was approximately 0.5 μM methylene blue.

FIG. 11 compares the kinetics of adsorption of methylene blue over a2-hour period from 100% plasma. Referring to FIG. 11, XAD-16 HP data isrepresented by open diamonds connected by dashed lines, the MN-200 datais represented by shaded triangles connected by solid lines, theXUS-43493 data is represented by open circles connected by dashed lines,and the fiberized resin containing XAD-16 is represented by shadedsquares connected by solid lines. As the data indicate, the XAD-16 HPand MN-200 gave the fastest adsorption kinetics, followed by XUS-43493.The slightly slower kinetics of the XUS-43493 may be a result of slowerwetting, as it was used in the dry state. Finally, the fiberized resincontaining XAD-16 had the slowest adsorption kinetics. This may haveresulted from poor contacting between the fiberized resin and plasmaduring the batch incubation, as a portion of the 14 cm² strip offiberized resin was not completely submersed in the plasma throughoutthe adsorption study, thereby reducing the effective contact areabetween the adsorbent and plasma.

The data indicate that non-psoralen pathogen-inactivating compounds likethe phenothiazine dyes can be removed from blood products using theresins and fiberized resin contemplated for use with the presentinvention.

Example 6 Removal of Acridine Compounds from Packed Red Blood Cells

This example is directed at the ability of a variety of different resinmaterials to remove acridine compounds from packed red blood cells(PRBCs). More specifically, the experiments of this example evaluate theremoval of the acridine compound, 5-[(β-carboxyethyl)amino]acridine,from PRBCs.

The chemical structures of several acridines are depicted in FIG. 12. Asindicated in FIG. 12, 9-amino acridine and5-[(β-carboxyethyl)amino]acridine are aminoacridines.

Resin Selectivity

Equilibrium adsorption of compound 5-[(β-carboxyethyl)amino]acridine wasstudied with several types of resins. The polymeric adsorbent resinsevaluated were Amberlite® XAD-2, XAD-4, XAD-7, and XAD-16 HP (Rohm andHaas); Purolite® MN-150, MN-170, MN-200, MN-300, MN-400, MN-500, andMN-600; and Dowex®XUS-43493 and XUS-40285 (Dow Chemical Co.). Inaddition, several Amberlite® anion exchange resins (IRA-958, IRA-900,IRA-35, IRA-410 and IRA-120; Rohm and Haas) and an Amberlite® weakcation exchange resin (DP-1; Rohm and Haas) were tested. Moreover,several charcoals were evaluated, including Hemosorba® AC (Asahi), PICAG277 and Norit A Supra (both commercially available from AmericanNorit). Finally, Porapak® RDx (Waters), a styrene vinyl pyrrolidonecopolymer which has affinity for nitro aromatic compounds, was alsotested.

Initially, an equilibrium adsorption study was performed with samples ofeach resin to evaluate capacity for 5-[(β-carboxyethyl)amino]acridineand adenine (6-aminopurine). Approximately 0.1 g of resin was weighedand transferred into a 6 mL polypropylene tube. A 5.0 mL aliquot of 25%plasma/75% Adsol® (Baxter) containing 100 μM of5-[(β-carboxyethyl)amino]acridine in distilled water was then added toeach tube. Cellular products such as red blood cells are typicallystored in a medium containing a low percentage of plasma (10-35%) with abalance of synthetic media; Adsol® is one example of a synthetic mediathat consists of adenine, dextrose, and mannitol in a saline solution.Of course, the present invention contemplates the use of concentrationsof acridines other than 100 μM in mixtures of plasma and other syntheticmedia. Next, the tubes were placed on a tumbling agitator and incubatedfor 3 hours at room temperature. Following incubation, aliquots of eachsample were removed for analysis of residual5-[(β-carboxyethyl)amino]acridine and adenine by HPLC.

For the HPLC procedure, each sample was diluted 2-fold with samplediluent (50% methanol, 25 mM KH₂PO₄, pH=3.5), and proteins and othermacromolecules were precipitated by incubating the samples at 4° C. for30 minutes. The samples were then centrifuged and the supernatant wasfiltered (0.2 μmeter) and analyzed on a C-18 reversed phase column (YMCODS-AM 4.6 mm×250 mm) by running a linear gradient from 75% solvent A(25 mM KH₂PO₄, pH=3.5), 25% B (methanol) to 80% B in 20 minutes. For5-[(β-carboxyethyl)amino]acridine removal, estimated capacities(μmole/g) at C_(f)=1 μM were determined from single adsorptionmeasurements with C_(o)=100 μM; the results are set forth in the secondcolumn of Table 6 (ND=not detectable). For adenine removal, estimatedcapacities (mmole/g) at C_(f)=1 mM were estimated from single adsorptionmeasurements with C_(o)=1.5 mM. The results are set forth in the thirdcolumn of Table 6 (ND=not detectable).

TABLE 6 Estimated 5-[(β- carboxyethyl)amino]acridine Adenine CapacityCapacity (μmole/g) (mmole/g) Resin at C_(f) = 1 μM at C_(f) = 1 mM XAD-20.1 0.00 XAD-4 3.2 0.02 XAD-7 0.1 0.01 XAD-16HP 4.3 0.03 XUS-43493 17.50.46 XUS-40285 6.8 0.27 MN-150 1.6 0.24 MN-170 4.2 0.43 MN-200 8.9 0.46MN-300 5.1 0.33 MN-400 4.4 0.22 MN-500 8.0 1.17 MN-600 5.8 0.36 IRA-9580.0 0.00 IRA-900 0.0 0.01 DP-1 0.0 0.00 IRA-35 0.0 0.01 IRA-120 4.7 0.41Hemosorba AC ND ND PICA G277 5.0 ND Norit A Supra ND ND Porapak RDx 0.10.01 IRA-410-D 0.0 0.00

While the use of any resin capable of adsorbing acridine compounds iscontemplated, preferred resins selectively adsorb5-[(β-carboxyethyl)amino]acridine over adenine and exhibit lowhemolysis. FIG. 13 plots the data for adenine capacity (y-axis) and5-[(β-carboxyethyl)amino]acridine capacity (x-axis) for various resins.As indicated by the data in Table 6 and FIG. 13, the Dowex® XUS-43493and Purolite® MN-200 resins had the highest5-[(β-carboxyethyl)amino]acridine capacity; moreover, when both high5-[(β-carboxyethyl)amino]acridine capacity and low adenine capacity wereconsidered, the Amberlite® XAD-16 HP performed well. The results fromthe in vitro experiments described in this example suggest that Dowex®XUS-43493 and the related resin Purolite® MN-200 are preferred resinsfor the removal of acridine compounds from PRBCs.

Example 7 Removal of Acridine Compounds from Packed Red Blood Cells withStyrene-Divinylbenzene Adsorbents

This example is directed at the ability of a variety of differentstyrene-divinylbenzene (styrene-DVB) adsorbents to remove aminoacridinecompounds from blood preparations. More specifically, the experiments ofthis example evaluate the removal of acridine orange and 9-aminoacridine (depicted in FIG. 12) from a plasma/Adsol® solution.

A. Experimental Procedures

For the experiments of this example, stock solutions (10 mM) of5-[(β-carboxyethyl)amino]acridine (Cerus) and acridine orange (Aldrich)were prepared in distilled water, and a stock solution (10 mM) of9-amino acridine (Aldrich) was prepared in ethanol. The5-[(β-carboxyethyl)amino]acridine was added to a solution containing 25%plasma/75% saline to achieve a final concentration of 100 μM, while theacridine orange and the 9-amino acridine compounds were added tosolutions containing 25% plasma/75% Adsol® (Baxter) Red CellPreservation Solution to achieve a final acridine concentration of 100μM.

The adsorbents utilized were Amberlite® XAD-16 HP (Rohm and Haas);Purolite® MN-200, and Dowex® XUS-43493 (Supelco). The water content ofeach adsorbent was determined by measuring the mass loss upon drying;the water content was corrected for so that the equivalent of 0.25 g dryof each adsorbent was used. Adsorbents were accurately weighed into 50mL Falcon tubes. Thirty (30) mL of the 25% plasma/75% Adsol® solutioncontaining 100 μM acridine was added to each tube containing adsorbent.The tubes were then placed on a rotator at room temperature, and 500 μLsamples of solution were removed at various times and stored for lateranalysis.

Samples were analyzed by HPLC for levels of residual acridine. Eachsample was diluted 2-fold with sample diluent (50% methanol, 25 mMKH₂PO₄, pH=3.5), and proteins and other macromolecules were precipitatedby incubating the samples at 4° C. for 30 minutes. The samples were thencentrifuged and the supernatant was filtered (0.2 μmeter) and analyzedon a C-18 reversed phase column (YMC ODS-AM 4.6 mm×250 mm) by running alinear gradient from 75% solvent A (25 mM KH₂PO₄, pH=3.5), 25% B(methanol) to 80% B in 20 minutes. Detection was by visible absorbanceusing a diode array detector set at 400 nm for 9-amino acridine, 490 nmfor acridine orange, and 410 nm for 5-[(β-carboxyethyl)amino]acridine.

B. Adsorption Kinetics

The kinetics of adsorption of 5-[(β-carboxyethyl)amino]acridine over a3-hour period from 25% plasma/75% saline were compared for Dowex®XUS-43493, Purolite® MN-200, and Amberlite® XAD-16 HP. FIG. 14A and FIG.14B both represent residual 5-[(β-carboxyethyl)amino]acridine as afunction of time, FIG. 14B presenting the data on a logarithmic scale.In FIG. 14A and FIG. 14B, the XAD-16 HP data is represented by shadedcircles connected by dashed lines, the MN-200 data is represented byshaded squares connected by dashed lines, and the XUS-43493 data isrepresented by shaded triangles connected by solid lines. As the dataindicate, the XUS-43493 and MN-200 gave the fastest adsorption kineticsand were nearly equivalent. The XAD-16 HP appears to have a lowercapacity for 5-[(β-carboxyethyl)amino]acridine.

FIG. 15 compares the adsorption kinetics for removal of 9-amino acridineand acridine orange from 25% plasma/75% Adsol® with Dowex® XUS-43493.Referring to FIG. 15, the shaded squares with the dashed lines represent9-amino acridine, and the shaded circles with the solid lines representacridine orange. As the data indicate, levels of 9-amino acridine wereundetectable beyond 3 hours. By comparison, the capacity of the Dowex®XUS-43493 for acridine orange was lower, which may be related to thepresence of two tertiary amino groups on acridine orange.

Example 8 Preparation of PRBCs Treated with a5-[(β-carboxyethyl)amino]acridine Derivative and Glutathione

Pools of PRBC were prepared from fresh ABO-matched whole blood. Theunits of whole blood were centrifuged at 3800 rpm for 5 minutes using aBeckman Sorvall RC3B centrifuge. The plasma was expressed into anotherclean bag using an expresser. The units of PRBC were pooled into a 3.0 Lsize Clintec Viaflex bag if more than one unit was needed. For each unitof PRBC, 94 mL of Erythrosol was added. The percentage of Hematocrit(HCT) was measured by filling a capillary tubing with the blood sampleand spinning it for 5 minutes. The hematocrit was determined to ensurethat it did not decrease below 55%. For each 100 mL of PRBC, 3.3 mL of12% glucose was added. The final percentage hematocrit was determined.PRBC (300 mL) was refilled into plastic containers PL146 (BaxterHealthcare). To the blood bags was added 6.0 mL of 150 mM glutathione toreach a 3.0 mM final concentration and 3.0 mL of 30 mM a5-[(β-carboxyethyl)amino]acridine derivative for a final concentrationof 300 μM. The PRBC mixture was agitated for 1 minute using a wristaction shaker (manufacturer). The 5-[(β-carboxyethyl)amino]acridinederivative and glutathione treated PRBCs were allowed to incubate atroom temperature overnight to allow break down of the5-[(β-carboxyethyl)amino]acridine derivative into5-[(β-carboxyethyl)amino]acridine.

Example 9 HPLC Assay for 5-[(β-carboxyethyl)amino]acridine in PRBC andPRBC Supernatant

The sample (100 μL) was diluted with 100 μL of saline and the resultingmixture was vortexed. To the solution was added 300 μL of 20 mM H₃PO₄ inCH3CN, and the mixture was vortexed for 15 sec. The sample wascentrifuged at 13,200 rpm for 5 minutes. The supernatant (200 μL) wasdiluted into 800 μL of cold 0.1 M HCl and vortexed. The sample wasfiltered into an autosampler vial using a 0.2 μm Gelman Acrodisc filter.The HPLC conditions were as follows: (Manufacturer and part no. forcolumn and general column.) Column=Zorbax SB-CN, 4.6 mm×150 mm, 3.5 μmparticles; guard column=(4.6 mm×12.5 mm, 5 μm particles (Mac ModAnalytical, Inc. (Chadds Ford, Pa.)); the mobile phase for A was 10 mMH₃PO₄ in HPLC water; the mobile phase for C was 10 mM H₃PO₄ inacetonitrile; temperature was 20° C.; sample volume was 100 μL; gradientconditions were as follows:

Flow Rate Time A (%) C (%) (mL/min) 0.00 90.0 10.0 1.0 5.28 77.0 23.01.0 10.00 40.0 60.0 1.0 11.00 90.0 10.0 1.0 16.00 STOP STOP STOPthe DAD settings were as follows:

Detection Detection Reference Reference Signal Wavelength BandwidthWavelength Bandwidth A 410 5 580 20 B 260 5 580 20

Example 10 HPLC Assay for Glutathione in PRBC and PRBC Supernatant

The sample was prepared as for the HPLC assay described above. The HPLCconditions were as follows: analytical column=YMC ODS-AM-303, 250 mm×4.6mm, 5 μm particle; guard column=Brownlee, 15×3.2 mm, 7 μm particle; themobile phase for A was 10 mM H₃PO₄ in HPLC water; the mobile phase for Cwas 10 mM H₃PO₄ in acetonitrile; the temperature was 15° C.; the samplevolume was 75 μL; the gradient conditions were as follows:

Flow Rate Time A (%) C (%) (mL/min) 0.00 95.0 5.0 0.5 6.00 95.0 5.0 0.58.00 10.0 90.0 1.0 9.00 95.0 5.0 1.0 20.00 STOP STOP STOPthe DAD settings were as follows:

Detection Detection Reference Reference Signal Wavelength BandwidthWavelength Bandwidth A 205 10 600 100

Example 11 Method of Screening Adsorbents

A test solution containing 25% plasma and 75% Erythrosol was used torepresent the supernatant from PRBCs. Erythrosol was prepared as twoseparate stock solution parts (solution C and solution D) that weresterilized separately. The final solutions were prepared by mixing equalvolumes of solution C and solution D.

Solution C Solution D 3.2 mM Adenine 53.2 mM Na citrate 2 H₂O 85 mMmannitol 5.4 mM NaH₂PO₄ 2 H₂O 100 mM glucose 38 mM Na₂HPO₄ 2 H₂O 6.2 mMHClThe plasma-Erythrosol solution was spiked with5-[(β-carboxyethyl)amino]acridine to a final concentration of 300 μM.Glutathione (reduced form, Sigma Chemical Co.) was added to the mixtureto obtain a final concentration of 3 mM. Samples of adsorbents (0.2-0.8g) were accurately weighed (±0.001 g) into tared 7 mL polypropylenevials with screw tops. Samples of adsorbent that required pre-wettingwere suspended in 70% ethanol. The adsorbent was allowed to settle andthe supernatant was removed. Residual ethanol was removed byresuspending the adsorbent in distilled water, allowing the adsorbent tosettle, and decanting the supernatant. Adsorbent masses were correctedfor water content when adsorption capacities were calculated. A 5.0 mLaliquot of plasma/Erythrosol was added to each tube. The tubes wereplaced on a rotator and tumbled at room temperature for 24 hours. Thevials were removed from the rotator and the adsorbent was allowed tosettle. A sample of the supernatant was removed from each tube. Sampleswere centrifuged to remove residual adsorbent. Samples were analyzed byHPLC to determine residual levels of 5-[(β-carboxyethyl)-amino]acridine.The reversed phase assay described above was used to determine residuallevels of glutathione. The results from the adsorbent screen are shownin Tables 7 and 8.

TABLE 7 Adsorbent Screen - Removal of 5-[(β-carboxyethyl)amino]acridinefrom 25% Plasma/75% Erythrosol. (“LOD” is limit of detection.) FinalCorrected Wetting Acridine Capacity Estimated Adsorbent Chemistry Mass(g) Required? C_(f) (μM) (μmole/g) K (L/g) Purolite MN-150 Weak base,PS- 0.108 No 1.4 13.5 9.7 DVB Purolite MN-170 Weak base, PS- 0.105 No0.4 14.0 38.4 DVB Purolite MN-200 Nonfunctional, 0.077 No 0.9 19.1 21.5PS-DVB Purolite MN-300 Weak Base, PS- 0.111 No 1.0 13.2 13.4 DVBPurolite MN-400 Strong base, PS- 0.102 No 1.1 14.3 13.0 DVB PuroliteMN-500 Strong acid, PS- 0.098 No 0.6 15.0 24.2 DVB Purolite MN-600 Weakacid, PS- 0.112 No 0.8 13.1 16.7 DVB Dowex Optipore L- Nonfunctional0.189 No 0.3 7.8 22.5 493 PS-DVB 46 A, 1100 m²/g Dowex Optipore L- Weakbase, PS- 0.245 No 0.5 6.0 11.2 285 DVB 25 A, 800 m²/g Amberlite XAD-2Nonfunctional 0.190 Yes 117.5 4.7 0.0 PS-DVB 90 A, 300 m²/g AmberliteXAD-4 Nonfunctional 0.118 Yes 1.6 12.4 7.7 PS-DVB 40 A, 725 m²/gAmberlite XAD-7 Polyacrylic ester 0.097 Yes 117.4 9.1 0.1 90 A, 450 m²/gAmberlite XAD-16 Nonfunct. 0.101 Yes 44.1 12.4 0.3 polystyrene 100 A,800 m²/g Hemosorba AC Activated 0.149 No LOD >9.8 >196.8 charcoalpHEMA-coated Duolite GT-73 Thiol-containing 0.144 No 1.0 10.2 10.2macroreticular Kieselguhr Diatomaceous 0.131 No 317.7 −0.9 0.0 earthGraver GL-711 Ground XUS- 0.134 No 1.9 10.9 5.8 43493 attached to fiberAmberlite IRA 900 Quat. Am. 0.067 No 239.1 4.1 0.0 macroreticular PS,Cl-form Amberlite IRA 35 Weak base 0.076 No 223.7 4.6 0.0 modified PSAmberlite Strong base gel 0.086 No 127.2 9.7 0.1 IRA 410 D PS Cl-formAmberlite IRA 958 Quat Am. 0.084 No 304.9 −0.6 0.0 macroreticularPolystyrene Cl- form Amberlite DP-1 Carboxylic 0.066 No 292.8 0.1 0.0macroreticular PS Amberlite IR-120 Sulfonic acid gel 0.079 No 1.4 18.613.1 PS H-form Diaion HPA 75 Quat. alkyl amine 0.091 No 97.2 10.8 0.1highly porous PS Duolite S-761 Phenol- 0.093 No 1.8 15.8 8.6formaldehyde modified PS Duolite A-7 Polyamine 0.079 No 189.0 6.6 0.0macroreticular PS, free base Amberlite IRA-68 Polyamine gel PS 0.083 No203.4 5.5 0.0 Amberlite IRA-458 Quat. Am. gel PS, 0.148 No 292.8 0.0 0.0Cl-form Amberlite IRA-958 Quat. Am. 0.139 No 296.3 −0.1 0.0macroreticular PS, Cl-form Ambersorb 563 Synthetic AC, 550 m²/g, 0.228No 0.4 6.4 17.5 high hydrophobicity Ambersorb 572 Synthetic AC, 0.267 NoLOD >5.5 >55.0 1100 m²/g, low hydrophobicity Ambersorb 575 Synthetic AC,800 m²/g, 0.258 No LOD >5.7 >56.9 mid hydrophobicity PICA G277 ACActivated charcoal 0.283 No LOD >5.2 >51.9 PICA NC506 AC Activatedcharcoal 0.266 No LOD >5.5 >55.3 PICAtif Med. AC Activated charcoal0.280 No LOD >5.3 >52.5 West VACO CX-S Activated charcoal 0.228 NoLOD >6.4 >64.4 Norit ASupra Activated charcoal 0.268 No LOD >5.5 >54.8Norti B Supra Activated charcoal 0.258 No LOD >5.7 >56.9 Norit Supra EActivated charcoal 0.266 No LOD >5.5 >55.2 Norit S51 AC Activatedcharcoal 0.237 No LOD >6.2 >61.9 Norit SX Ultra Activated charcoal 0.203No LOD >7.2 >72.2 Chemviron Activated charcoal 0.233 No LOD >6.3 >63.1Norit CN1 Activated charcoal 0.261 No LOD >5.6 >56.4 Norit G60 Activatedcharcoal 0.230 No LOD >6.4 >64.0 Norit ROX, O, 8 Activated charcoal0.246 No LOD >6.0 >59.8 Norti Darco Activated charcoal 0.219 NoLOD >6.7 >67.3 (20 × 50) PICAtif Medicinal Activated charcoal 0.129 NoLOD >11.4 >113.8 Davison Silica Unmodified silica 0.245 No 77.0 4.4 0.1Grade 15 Davison Silica Unmodified silica 0.255 No 140.1 3.0 0.0 Grade636 BioRad AG501- Mixed bed ion 0.083 No 119.4 10.5 0.1 X8(D) exchangeAmberlite 200 Sulfonic acid 0.080 No 46.5 15.5 0.3 macroret. PS, Na-form MP-3 (C-18) SPE Sulfonated C-18 0.239 Yes 1.7 6.1 3.7 media resinAlltech C-18 SPE C-18 modified 0.234 Yes 64.4 4.9 0.1 silica BioRadt-Butyl C-4 modified 0.213 Yes 163.9 3.1 0.0 HIC polymethacrylate BakerC-18 SPE C-18 modified 0.263 Yes 94.9 3.8 0.0 silica Waters Sep Pak C-C-18 modified 0.190 Yes 81.3 5.6 0.1 18 silica Baker C-4 SPE C-4modified 0.232 Yes 178.3 2.5 0.0 silica Waters Bondapak C-8 modified0.213 Yes 156.9 3.2 0.0 C-8 silica Waters Bondapak C-4 modified 0.228Yes 251.0 0.9 0.0 C-4 silica Amberchrom cg- Polystyrene 0.192 Yes 79.35.6 0.1 161 xcd 150 A, 900 m²/g Amberchrom cg- Polystyrene 0.176 Yes243.7 1.4 0.0 1000 sd 1000 A, 250 m²/g Amberchrom cg- Polystyrene 0.135Yes 172.1 4.5 0.0 300 md 300 A, 700 m²/g Amberchrom cg-71Polymethacrylate 0.189 Yes 156.4 3.6 0.0 md 250 A, 500 m²/g WatersPorapak PS-vinyl 0.125 Yes 307.8 −0.6 0.0 RDx pyrrolidone porousadsorbent CUNO Delipid Resin-modified 0.663 No 245.8 0.4 0.0 Mediacellulose CUNO Weak base 0.361 No 280.8 0.2 0.0 DEAE media modifiedcellulose Sigma DE Diatomaceous 0.179 No 317.6 −0.7 0.0 earth DiaionSP-850 Polystyrene 0.236 Yes 1.4 6.2 4.4 38 A, 1000 m²/g Diaion SP-207Brominated PS 0.191 No 211.1 2.2 0.0 105 A, 650 m²/g Diaion HP-2MGPolymethacrylate 0.258 Yes 182.6 2.2 0.0 170 A, 500 m²/g Diaion HP-20Polystyrene 0.175 Yes 107.4 5.3 0.0 260 A, 500 m²/g Amberlite 1180Polystyrene-DVB 0.094 Yes 9.9 15.1 1.5 300 A, 600 m²/g Amberilte 1600Polystyrene-DVB 0.208 Yes 73.0 5.3 0.1 Amberlite XAD- Polystyrene-DVB0.172 Yes 61.0 6.8 0.1 2000 42 A, 580 m²/g Amberlite XAD-Polystyrene-DVB 0.203 Yes 173.0 3.0 0.0 2010 280 A, 660 m²/g Dowex XUS-Polystyrene-DVB 0.213 Yes 102.6 4.5 0.0 40323 100 A, 650 m²/g WhatmanDE-52 Weak base 0.284 No 177.5 2.0 0.0 modified cellulose Whatman CM-32Weak acid 0.294 No 268.7 0.4 0.0 modified cellulose Whatman QA-52 Strongbase 0.264 No 285.2 0.2 0.0 modified cellulose Whatman SE-53 Strong acid0.238 No 294.3 0.0 0.0 modified cellulose Pharmacia Q Seph Strong base0.111 No 267.8 1.2 0.0 FF modified agarose Pharmacia S Seph Strong acid0.112 No 267.0 1.2 0.0 FF modified agarose Toyopearl QAE- Wead base0.118 No 266.8 1.2 0.0 550 C modified agarose Toyopearl ButylHydrophobic (C- 0.112 Yes 232.8 2.7 0.0 650-M 4) modified methacrylateToyopearl SP- Strong acid 0.111 No 230.2 2.9 0.0 550C modifiedmethacrylate Toyopearl CM- Weak acid 0.118 No 249.0 1.9 0.0 650 Mmodified methacrylate Toyopearl Weak base 0.112 No 260.4 1.5 0.0DEAE-650M modified methacrylate Toyopearl Super Q Strong base 0.111 No263.2 1.4 0.0 650 C modified methacrylate

TABLE 8 Adsorbent Screen - Removal of Glutathione from 25% Plasma/75%Erythrosol Estimated GSH Final GSH Estimated Capacity at HPLC Area Conc.Capacity at C_(f) C_(f) = 30 μM Adsorbent (mAU*sec) (μM) (μmole/g)(μmole/g) Purolite MN-150 336.1 2375 28.9 0.4 Purolite MN-170 386.7 273212.8 0.1 Purolite MN-200 445.5 3147 −9.6 −0.1 Purolite MN-300 356.7 252021.7 0.3 Purolite MN-400 297.0 2099 44.1 0.6 Purolite MN-500 392.8 277511.5 0.1 Purolite MN-600 392.4 2773 10.1 0.1 Dowex XUS-43493 428.5 3027−0.7 0.0 Dowex XUS-40285 251.6 1778 24.9 0.4 Amberlite XAD-2 420.8 29730.7 0.0 Amberlite XAD-4 449.0 3172 −7.3 −0.1 Amberlite XAD-7 448.0 3165−8.5 −0.1 Amberlite XAD-16 412.2 2912 4.4 0.0 Hemosorba AC 7.9 56 98.653.3 Duolite GT-73 379.3 2680 11.2 0.1 Kieselguhr 471.3 3330 −12.6 −0.1Graver GL-711 466.2 3294 −11.0 −0.1 Amberlite IRA 900 441.1 3116 −8.6−0.1 Amberlite IRA 35 434.4 3069 −4.5 0.0 Amberlite IRA 410 D 413.0 29184.8 0.0 Amberlite IRA 958 446.2 3152 −9.1 −0.1 Amberlite DP-1 433.3 3061−4.6 0.0 Amberlite IRA-120 388.6 2745 16.2 0.2 Diaion HPA 75 447.3 3160−8.8 −0.1 Duolite S-761 312.9 2211 42.6 0.6 MP-3 428.0 3024 −0.5 0.0Alltech C-18 SPE 411.3 2906 2.0 0.0 Duolite A-7 444.2 3138 −8.7 −0.1 AmbIRA-68 443.5 3133 −8.1 −0.1 Amb IRA-458 460.6 3254 −8.6 −0.1 Amb IRA-958426.3 3012 −0.4 0.0 Ambersorb 563 435.2 3075 −1.6 0.0 Ambersorb 572 0.0LOD >56.1 >167.9 Ambersorb 575 0.0 LOD >58.1 >173.8 PICA G277 AC 0.0LOD >53.0 >158.4 PICA NC506 AC 0.0 LOD >56.4 >168.7 PICAtif Med. AC 0.0LOD >53.6 >160.4 West VACO CX-S 0.0 LOD >65.7 >196.5 Norit A Supra 0.0LOD >55.9 >167.2 Norti B Supra 0.0 LOD >58.1 >173.7 Norit Supra E 0.0LOD >56.3 >168.5 Norit S51 AC 0.0 LOD >63.2 >189.0 Norit SX Ultra 0.0LOD >73.7 >220.5 Chemviron 0.0 LOD >64.4 >192.6 Norit CN1 0.0LOD >57.5 >172.1 Norit G60 0.0 LOD >65.3 >195.4 Norit ROX, O, 8 0.0LOD >61.0 >182.4 Norti Darco (20 × 50) 0.0 LOD >68.6 >205.3 PICAtifMedicinal 0.0 LOD >116.2 >347.4 Whatman 150A Silica 448.8 3171 −3.6 0.0Davison Silica Grade 15 443.5 3133 −2.7 0.0 Davison Silica Grade 636423.9 2995 0.1 0.0 BioRad AG501-X8(D) 445.6 3148 −8.9 0.1 Amberlite 200458.1 3237 −14.8 0.1 BioRad t-Butyl HIC 484.0 3419 −9.8 −0.1 Baker C-18SPE 427.7 3022 −0.4 0.0 Waters Sep Pak C-18 422.2 2983 0.4 0.0 Baker C-4SPE 429.8 3036 −0.8 0.0 Waters Bondapak C-8 405.5 2865 3.2 0.0 WatersBondapak C-4 390.8 2761 5.2 0.1 Amberchrom cg-161 xcd 414.8 2931 1.8 0.0Amberchrom cg-1000 sd 373.5 2639 10.2 0.1 Amberchrom cg-300 md 396.22799 7.4 0.1 Amberchrom cg-71 md 396.4 2800 5.3 0.1 Waters Porapak RDx449.9 3179 −7.1 −0.1 CUNO Delipid Media 452.5 3197 −1.5 0.0 CUNO DEAEmedia 213.7 1510 20.6 0.4 Sigma Diatomaceous 473.9 3348 −9.7 −0.1 EarthDiaion SP-850 424.1 2996 0.1 0.0 Diaion SP-207 471.6 3332 −8.7 −0.1Diaion HP-2MG 429.7 3036 −0.7 0.0 Diaion HP-20 422.3 2984 0.5 0.0 Amb1180 441.7 3120 −6.4 −0.1 Amberilte 1600 426.6 3014 −0.3 0.0 AmberliteXAD-2000 407.4 2879 3.5 0.0 Amberlite XAD-2010 423.0 2988 0.3 0.0 DowexXUS-40323 372.7 2633 8.6 0.1 Whatman DE-52 384.7 2718 5.0 0.1 WhatmanCM-32 514.8 3637 −10.8 −0.1 Whatman QA-52 398.1 2813 3.5 0.0 WhatmanSE-53 458.0 3236 −4.9 0.0 Pharmacia Q Seph FF 377.6 2668 15.0 0.2Pharmacia S Seph FF 390.5 2759 10.8 0.1 Toyopearl QAE-550 C 395.1 27928.8 0.1 Toyopearl Butyl 650-M 372.9 2635 16.2 0.2 Toyopearl SP-550C404.2 2856 6.5 0.1 Toyopearl CM-650M 394.0 2784 9.2 0.1 ToyopearlDEAE-650M 392.3 2772 10.2 0.1 Toyopearl Super Q 650C 384.6 2717 12.8 0.1

Example 12 Adsorption Capacities for Adsorbents

Adsorption isotherm experiments were carried out to determine theadsorptive capacities (μmole 5-[(β-carboxyethyl)-amino]acridine/gadsorbent) for various types of adsorbents. FIG. 17 shows adsorptionisotherms obtained for several Ambersorbs as compared to the adsorptionisotherm for Purolite MN-200. Adsorption studies were performed in 25%plasma/75% Erythrosol solutions containing 0.2-3 mM5-[(β-carboxyethyl)amino]acridine and 0.6-10 mM GSH. Samples wereagitated for 24 hours at room temperature. Calculations using theadsorption capacity from Table 7 (22 μmole/g) determined thatapproximately 4 g of Purolite MN-200 would be required to reduce thelevel of 5-[(β-carboxyethyl)-amino]acridine in a 300 mL unit of PRBCfrom 300 μM to a final level of 1 μM. Less than 1 g of Ambersorb 572(130 mmole/g) would be required to achieve comparable removal. A similarcalculation estimated that less than 1 g of Ambersorb 572 would berequired to reduce the level of GSH in a 300 mL unit of PRBC from 6 mMto a final level of 500 μM in the 150 mL of supernatant (50% HCT).

Example 13 Long Term Removal of Breakdown Products

This experiment examined 5-[(β-carboxyethyl)-amino]acridine and GSHlevels in PRBCs from which a fiberized PICA G-277 activated carbondevice (AQF, 7.3 g, 500 m²/g) was removed after 24 hours of exposure.This study was conducted in parallel with studies where the PRBCs hadcontinued device exposure. FIG. 18 shows that the concentrations of5-[(β-carboxyethyl)-amino]acridine and GSH in the supernatant sampleswere considerably higher in the absence of a device over storage timesof 1 to 4 weeks.

The concentration of 5-[(β-carboxyethyl)-amino]acridine was reduced to 5μM in initially shaken PRBCs after 35 days of storage in the presence ofa removal device (MN-200). This indicates that5-[(β-carboxyethyl)-amino]acridine removal does occur in static storageconditions at 4° C.

Example 14 Effect of Enclosure Material (Membrane, Woven, Non-Woven) onan IAD

The use of an enclosure material surrounding the adsorbent media wasinvestigated for the primary purpose of particle retention. The primarypurpose is particle retention. However, membranes can enhancehemocompatibility of the devices by preventing contact between the RBCsand membranes. Membranes can easily be modified with hydrophilicpolymers (PEO, PEG, HPL) to enhance hemocompatibility without alteringfunction. Approximately 10 g of fiberized Pica G277 activated carbonmedia (AQF 500 g/m²) was surrounded by a heat-sealed membrane, wovenpolyester, or non-woven polyester material. PRBC units (300 mL) weredosed with 300 μM of a degradable 5-[(β-carboxyethyl)amino]acridinederivative and 3 mM GSH, held at room temperature for 20 hours on aplatelet shaker, and then transferred to IADs. Concentration of5-[(β-carboxyethyl)-amino]acridine was monitored over 24 hours. FIG. 19shows 5-[(β-carboxyethyl)-amino]acridine levels in the supernatant of300 mL PRBC units exposed to IADs consisting of fiberized Pica G277activated carbon (500 g/m²) and enclosed by a membrane, woven, ornon-woven material. PRBCs (Erythrosol, glucose, 62% HCT) were dosed with300 μM of a degradable 5-[(β-carboxyethyl)amino]acridine derivative and3 mM GSH, and agitated on a platelet shaker at room temperature prior totransfer to the IADs. PRBC-containing IADs were agitated at roomtemperature for 24 hours.

These studies indicate that the Tetko woven enclosure shows the fastestremoval kinetics for 5-[(β-carboxyethyl)-amino]acridine over 24 hours.Final levels achieved for all enclosure materials after 2 weeks weresimilar, with 5-[(β-carboxyethyl)-amino]acridine concentrationsdecreasing to approximately 2 μM after 1 day, but rising back to 10 μMnear day 8.

Example 15 Effect of the Compound Adsorption Device on Red Blood CellFunction

Indicators of red blood cell function were monitored over the course of5-[(β-carboxyethyl)-amino]acridine and GSH removal experiments forvarious device configurations. Parameters measured included percentagelysis, ATP and K⁺ concentration. Table 9 shows that ATP concentrationswere generally not affected by the presence of a compound removaldevice: The decrease in ATP concentrations was approximately the samefor Control (no IAD) as for the MN-200 or Pica G277 devices. Levels ofK⁺ in PRBCs were found to increase with time. The temperature at whichremoval occurred did not influence K⁺ levels in PRBC units exposed tocompound removal devices over 20 days. Where the time period of exposurewas extended to 35 days, however, the rate of increase of K⁺ in PRBCsvaried with the type of adsorbent, with final levels achieved of 40 and45 mmol/L for MN-200 and PICA G-277 devices, respectively, compared tothe no device control at 39 mmol/L. The percentage of red blood cellslysed in device-exposed and no-device control PRBC units has generallybeen found to be between 0.1 and 1% after 24 hours. As shown in Table 10and Table 11 and graphed in FIGS. 20 and 21, the % lysis variedsignificantly with the type of adsorbent used. Table 10 shows lysisvalues obtained for PRBCs exposed to two types of immobilized adsorptiondevices over 35 days. Table 11 shows lysis values obtained for PRBCsexposed to loose adsorbent particles enclosed in a woven polyester meshover 42 days. A wrist action shaker was used in dosing all PRBC unitsfor 1 minute, after which the PRBCs were in a static condition for 4hours at room temperature. The devices were held at room temperature for24 hours on a platelet shaker, after which they were in a staticcondition at 4° C. for the duration of the study. The immobilizationMN-200 showed lower hemolysis levels than immobilized PICA G-277, whilethe Ambersorb synthetic carbonaceous adsorbent showed one of the lowesthemolysis levels upon comparison of the loose particle adsorbents. TheMN-200 IAD showed lower hemolysis than the same non-immobilizedadsorbent. Similar observations have been observed for other IADs ascompared to the same non-immobilized adsorbent.

TABLE 9 ATP Concentration (μmol/dL) in PRBCs over time Time Control (noIAD) MN-200 IAD PICA G277 IAD 0 hr 76 78 79 4 hr 80 79 82 8 hr 82 82 8124 hr 83 83 86 8 days 73 70 78 22 days 50 42 50 35 days 25 23 28

TABLE 10 Percent Lysis in PRBCs (56% HCT) exposed to different IADs overtime Time Control (No IAD) MN-200 IAD PICA G277 IAD 0 hr 0.07 0.09 0.134 hr 0.11 0.14 0.37 8 hr 0.12 0.18 0.53 24 hr 0.13 0.21 0.62 8 days 0.180.22 0.67 22 days 0.22 0.24 0.77 35 days 0.34 0.33 0.89

TABLE 11 Percent lysis in PRBCs (55% HCT) exposed to different looseparticle adsorbents enclosed in woven mesh. Purolite Darco Control MN-Hemosorba Ambersorb AC Pica Time no IAD 200 CH-350 572 20 × 50 G277 0 hr0.10 0.07 0.08 0.07 0.06 0.07 4 hr 0.07 0.09 0.38 0.10 0.08 0.13 8 hr0.09 0.17 0.54 0.12 0.12 0.18 24 hr  0.11 0.39 1.06 0.16 0.13 0.92 Day14 0.12 0.48 1.11 0.22 0.13 0.40 Day 21 0.15 0.59 1.24 0.29 0.17 0.57Day 28 0.22 0.60 1.52 0.40 0.26 0.74 Day 35 0.30 0.78 1.85 0.55 0.411.05 Day 42 0.53 1.08 2.72 0.86 0.79 1.60

The critical nature of the adsorbent particle with respect to themaintenance of red blood cell function is illustrated. A comparativestudy of the effects of five different adsorbents on red blood cellhemolysis is presented. Ambersorb 572 produced only 0.16% lysis in PRBCs(55%) after a 24 hour exposure, while Darco AC (Norit Americas, Inc.(Atlanta, Ga.) produced 0.13% lysis. Those adsorbents were significantlybetter at minimizing hemolysis of red blood cells than the PuroliteMN-200, Hemosorba CH-350 and Pica G277 adsorbents.

Table 12 shows a comparative study where supernatants from red bloodcell samples containing glutathione and5-[(β-carboxyethyl)amino]acridine were contacted with a number ofdifferent adsorbents. Activated carbon adsorbents were the only type ofadsorbent that was capable of substantially reducing the concentrationsof both 5-[(β-carboxyethyl)-amino]acridine and glutathione. Both naturalactivated carbons (Norit and PICA) and synthetic activated carbons(Ambersorb) proved to be effective at compound reduction.

TABLE 12 Properties of Several Different Adsorbents GSH Cap{circumflexover ( )} Acridine Cap{circumflex over ( )} (μmole/g) (μmole/g)Adsorbent Manufacturer Description^(@) C_(f) = 500 μM C_(f) = 1 μMMN-200 Purolite Hypercrosslinked     0.0*  17⁺ macroreticular PS-DVB,200-1200 μm particles 1100 m²/g Optipore L-493 Dow ChemicalHypercrosslinked     0.0*  22* macroreticular PS-DVB, 300-840 μmparticles 1100 m²/g, 46 Å avg. pore diameter Duolite GT-73 Rohm & HaasMacroporous adsorbent     1.7*  10* with thiol functional groups Norit ASupra Norit Americas, Steam lignite AC, >2790* 560⁺ Inc. 2000 m²/g, 97%< 150 μm particles Picatiff Med. PICA Powdered AC from >2670* >53*coconut husk, 2000 m²/g, 8-35 μm particles Norit ROX 0.8 NoritSteam-activate peat AC, >3040* >60* extruded 900 m²/g, 840-1000 μmcylinders Ambersorb 572 Rohm & Haas Synthetic AC from >2800* 134⁺sulfonated PS, 1100 m²/g, 300-840 μm particles Microporous (ca. 50%pores < 20 Å) G-277 PICA Granular activated >2640* >52* carbon fromcoconut husk, ^(@)PS-DVB = polystyrene-divinyl benzene, AC = activatedcarbon {circumflex over ( )}Values listed as “>” were singlemeasurements with residual levels below the assay LOD *Estimated fromsingle-point adsorption studies in 25% plasma, 75% Erythrosol.⁺Estimated from multi-point adsorption isotherms in 25% plasma, 75%Erythrosol.

Example 16

Fiberized media consisting of Dowex Optipore L-493 attached to anonwoven polyester fiber matrix (Hoechst-L493) has been manufactured bythe AQF division of Hoechst Celanese (Charlotte, N.C.). The performanceof this adsorbent media in a batch removal device for platelets wasevaluated.

Platelet Preparation

Single donor apheresis platelet units containing 3.5-4.5×10¹¹ plateletsin 300 mL of 35% autologous plasma, 65% PAS III were obtained from theSacramento Blood Bank Center. 4′-(4-Amino-2-oxa)butyl-4,5′,8-trimethylpsoralen (Baxter Healthcare) was added to each platelet unit to achievea final concentration of 150 μM. Platelet units (4-5) were pooled in asingle PL-2410 plastic container and thoroughly mixed. The platelet poolwas evenly divided into 4-5 1 L PL2410 plastic containers eachcontaining approximately 300 mL of the platelet mixture. Units werephotochemically treated with 3.0 J/cm² UV-A and transferred into theappropriate removal device for the study. All experiments included acontrol platelet unit which was photochemically treated (150 μMpsoralen, 3.0 J/cm² UVA) but was not contacted with a removal device.

Device Preparation

Standard removal devices containing 2.5 g of Dowex XUS 43493 wereprepared by Baxter Healthcare Corporation (Lot FX1032 D96F20042R).Experimental IADs were prepared with Hoechst-L493 media (HoechstCelanese Corp.) that was supplied as roll stock. Media was measured andcut to give the appropriate adsorbent mass for each IAD (5.0 g, and 7.5g). The cut media was placed in pouches constructed by impulse welding30 μm polyester mesh (Tetko). Mesh pouches containing the media wereautoclaved (121° C., 20 min) and placed in sterile PL 2410 plasticcontainers. Alternatively, mesh pouches were placed in PL 2410containers, the containers were sealed, and the entire assembly wassterilized by gamma-irradiation to 2.5 MRad (SteriGenics). Excess airwas manually evacuated from devices using a syringe prior to transfer ofthe photochemically treated platelets.

Adsorption Kinetics

Following photochemical treatment with4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen+UVA, the plateletmixtures were transferred to PL2410 plastic containers with removaldevices. The devices were placed on a standard platelet incubator(Helmer) and agitated at 70 cycles/min at 22° C. Samples of the plateletmixture were removed at 1 hour intervals for the first 8 hours ofstorage. These samples were stored at 4° C. and later analyzed forresidual 4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen by HPLCanalysis. The assay involves an initial sample preparation which lysesthe platelets and solubilizes the4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen and freephotoproducts. The supernatant from the sample preparation is analyzedon a C-18 reverse phase column with a gradient of increasing methanol inKH₂PO₄ buffer.

In Vitro Platelet Function

Platelet mixtures were agitated in contact with the removal devices for7 days. In one study, the platelet mixture was contacted with the IADfor 24 hours and transferred to sterile 1 L PL2410 plastic containersusing a sterile tubing welder (Terumo SCD 312). Platelets were placedback in the platelet incubator and stored for the remainder of the 7 daystorage period.

Following 5 or 7 days of storage, the platelet count and pH weredetermined for each platelet unit. The pH and pO2/pCO2 was measuredusing a CIBA-Corning model 238 Blood Gas Analyzer. The platelet count ofeach sample was determined using a Baker 9118+ Hematology analyzer.

Several assays were performed to evaluate in vitro platelet function.The shape change of platelet samples was monitored using a Chrono-Logmodel 500 VS whole blood aggregometer. Shape change was quantified asthe ratio of maximum change in light transmission following addition ofADP relative to the change in light transmission following addition ofEDTA.

The response of platelets to hypotonic stress was evaluated by theHypotonic Shock Response (HSR) assay. The change in light transmissionfollowing addition of a hypotonic solution was measured using aChrono-Log model 500 VS whole blood aggregometer. Data is reported aspercent recovery from the hypotonic stress two minutes after absorbancereached its minimum value.

The ability of platelets to aggregate in response to ADP/collagenagonist was indicated by change in optical transmission as measured by aChrono-Log model 500 VS whole blood aggregometer.

The status of the platelets was evaluated by scoring the platelet.Samples were blinded and morphology scores of 100 platelets were totaledfor each sample. The highest possible score is 400 (Disc=4,intermediate=3, sphere=2, dendrite=1, balloon=0).

Platelet activation as indicated by expression of p-selectin (GranularMembrane Protein, GMP-140) was measured. CD62 antibody was used for thetest sample, mouse control IgG1 was used for the negative control, andCD62 antibody with phorbal myristate acetate (PMA) was used for thepositive control. Samples were analyzed on a FACScan (Becton Dickinson).The percent of activation that is reported is relative to the positivecontrol and is the difference between the test value and negativecontrol value.

Adsorption Kinetics

The impact of incorporating the adsorbent beads into a fiber matrix wasinvestigated. Platelet units containing 4.0×10¹¹ platelets in 300 mL of35% plasma, 65% PAS III were treated with 150 μM4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen+3.0 J/cm² UVA. IADswere prepared from Hoechst-L493 with an adsorbent loading of 450 g/m².The IADs were sterilized by gamma irradiation. Following treatment withpsoralen+UVA, the platelets were transferred to the removal devices.Samples were removed at 1 hour intervals and analyzed for residual4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen by HPLC. FIG. 22compares the kinetics for removal of4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen for IADs containing5.0 g and 7.5 g Hoechst-L493 media to that of a standard removal devicecontaining 2.5 g of loose beads. IADs contained either 5.0 gHoechst-L493 (triangles), 7.5 g of Hoechst-L493 media (squares), or 2.5g of loose Dowex L493 adsorbent beads (circles). The Hoechst-L493 mediacontained adsorbent beads at a loading of 450 g/m².

The kinetics of 4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralenadsorption were slower for the Hoechst media when compared to an equalmass of loose adsorbent beads. The removal device containing 2.5 g ofloose beads achieved the lowest levels of residual4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen at short times (1 hr).However, at longer times, the IADs containing 7.5 g and 5.0 g ofHoechst-L493 media performed better. In this study the adsorptionkinetics were relatively rapid with all three removal devices achievingthe target of <0.5 μM residual 4′-(4-amino-2-oxa)butyl-4,5′,8-trimethylpsoralen in under 4 hours of contact.

Note that at long times (6-8 hr) the Hoechst-L493 IADs which contained ahigher mass of adsorbent achieved lower levels of residual4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen. This observationsuggests that the slower adsorption kinetics for the Hoechst-L493 IADsis a result of mass transport limitations (fluid flow vs. diffusion) andis not a result of loss of functional adsorption area due to fiberattachment. Additional studies indicate that Hoechst-L493 media withlower levels of adsorbent loading (200-300 g/m²) allows fluid topenetrate the media more readily resulting in faster kinetics ofadsorption. Previous studies (data not shown) with Hoechst mediacontaining Amberlite XAD-16 adsorbent at a loading of 160 g/m² indicatedthat adsorption kinetics were not affected by incorporation into a fibermatrix at a low level of loading.

Platelet Yield and In Vitro Platelet Function

Data from two separate studies are presented in this section. Theplatelet units within each study were derived from a single pool so thatthe effect of the IAD media format, adsorbent mass, and contact timecould be clearly evaluated.

The first study evaluated the effect of fiberization (Hoechst media) onyield and function of the platelets following extended contact (5 and 7days). A total of four platelet units that were derived from a singlepool were used in this study. The no-IAD control unit was treated withpsoralen+UVA. Two of the platelet units were contacted with 5.0 gHoechst-L493. One unit was contacted with the IAD for 24 hours beforebeing transferred to an empty PL 2410 storage container. The other unitremained in contact with the IAD for the duration of the study. TheHoechst-L493 IADs were sterilized with steam (120° C., 20 min). Thestandard removal device (2.5 g loose Dowex XUS-43493), which wasobtained from Baxter, was sterilized by gamma-irradiation. Note that theplatelets were not transferred away from the standard removal devicefollowing 8-16 hour contact as is typically the practice with the devicethat utilizes loose adsorbent particles. Results from platelet countsand in vitro function following 5 and 7 days of storage are summarizedin Table 13A and Table 13B respectively.

TABLE 13A (Day 5) Comparison of Hoechst-L493 Fiberized Media to StandardRemoval Device Platelet Yield and In Vitro Function following 5-DayStorage Platelet Count Yield Sample (×10¹¹/300 mL) (%) pH pCO₂ pO₂Control (+PCT-RD) 3.50 ± 0.11 100 6.91 18 120 5.0 g Hoechst/L-493 3.41 ±0.06 97 ± 4 6.95 21 95 Transfer at 24 hr 5.0 g Hoechst/L-493 3.33 ± 0.0895 ± 4 6.93 24 87 No Transfer 2.5 g Dowex Optipore 2.60 ± 0.07 74 ± 37.04 13 146 L-493 Baxter Lot FX1032 D96F20042R No Transfer Shape SampleChange HSR Aggregation Morphology GMP-140 Control (+PCT- 0.83 ± 0.260.31 ± 0.08 69 ± 4 273 67.2 IAD) 5.0 g Hoechst/L-493 0.90 ± 0.02 0.43 ±0.04 83 ± 0 268 61.5 Transfer at 24 hr 5.0 g Hoechst/L-493 0.83 ± 0.120.40 ± 0.01 80 ± 2 280 58.9 No Transfer 2.5 g Dowex 0.24 ± 0.04 0.38 ±0.02 47 ± 3 240 71.3 Optipore L-493 Baxter Lot FX1032 D96F20042R NoTransfer

TABLE 13B (Day 7) Comparison of Hoechst-L493 Fiberized Media to StandardRemoval Device Platelet Yield and In Vitro Function following 7-DayStorage Platelet Count Yield Sample (×10¹¹/300 mL) (%) pH pCO₂ pO₂Control (+PCT- 3.45 ± 0.06 100 6.97 13 126 IAD) 5.0 g Hoechst/L- 3.28 ±0.22 95 ± 7 6.90 18 105 493 Transfer at 24 hr 5.0 g Hoechst/L- 3.11 ±0.15 90 ± 5 6.88 21 100 493 No Transferred 2.5 g Dowex 2.29 ± 0.07 66 ±2 7.04 9 161 Optipore L-493 Baxter Lot FX1032 D96F20042R No TransferShape Sample Change HSR Aggregation Morphology GMP-140 Control (+PCT-0.54 ± 0.05 0.25 ± 0.03 48 ± 0  284 80.1 IAD) 5.0 g Hoechst/L- 0.76 ±0.02 0.64 ± 0.16 86 ± 11 267 72.7 493 Transfer at 24 hr 5.0 g Hoechst/L-0.67 ± 0.00 0.29 ± 0.04 79 ± 7  280 66.1 493 No Transfer 2.5 g Dowex0.31 ± 0.09 0.28 ± 0.06 29 ± 11 261 77.1 Optipore L-493 Baxter LotFX1032 D96F20042R No Transfer

The platelet yields for the Hoechst-L493 IADs (5.0 g) were substantiallybetter than the yield for the standard removal device (2.5 g). Losses of<10% were achieved for 7 days of storage in continuous contact with theHoechst-L493 IAD (5.0 g). Both platelet units that were treated with theHoechst-L493 IADs displayed better performance in the shape change,aggregation, and GMP-140 assays than the no-IAD control. The plateletsthat remained in contact with the Hoechst-L493 IAD (5.0 g) for theentire 5 days showed comparable in vitro function to the platelets thatwere transferred after 24 hours of contact. Interestingly, the plateletsthat remained in contact with the Hoechst-L493 IAD performed better inthe GMP-140 assay. The difference in performance between the two waseven larger after 7 days. This observation suggests that contact withthe IAD decreases the rate of p-selectin expression by platelets duringstorage.

The second study looked at IADs containing 5.0 g and 7.5 g ofHoechst-L493 media to determine if there was a significant decrease inplatelet yield or in vitro function with a higher mass of media. In thisstudy, the Hoechst-L493 IADs were sterilized by gamma irradiation. Astandard removal device (2.5 g Dowex XUS-43493) was included in thestudy. Platelets remained in contact with the removal devices for theentire duration of the study. Results from platelet counts and in vitrofunction following 5 and 7 days of storage are summarized in Table 14Aand Table 14B respectively.

TABLE 14A (Day 5) Evaluation of Gamma Sterilized Hoechst-L493 FiberizedMedia Platelet Yield and In Vitro Function following 5-Day Storage (NoTransfer) Platelet Count Yield Sample (×10¹¹/300 mL) (%) PH pCO₂ pO₂Control (+PCT- 3.96 ± 0.19 100 6.89 25 88 IAD) 5.0 g Hoechst/L-493 3.68± 0.16 93 ± 6 6.89 27 70 7.5 g Hoechst/L-493 3.44 ± 0.11 87 ± 5 6.87 2684 2.5 g Dowex 2.85 ± 0.13 72 ± 5 7.04 13 145 Optipore L-493 Baxter LotFX1032 D96F20042R Shape Sample Change HSR Aggregation Morphology GMP-140Control (+PCT- 0.56 ± 0.12 0.26 ± 0.04 73 ± 1 289 65.1 IAD) 5.0 gHoechst/L-493 0.45 ± 0.04 0.40 ± 0.00 76 ± 2 278 54.5 7.5 gHoechst/L-493 0.48 ± 0.06 0.25 ± 0.01 73 ± 2 290 55.4 2.5 g Dowex 0.04 ±0.06 0.30 ± 0.01 38 ± 3 232 62.8 Optipore L-493 Baxter Lot FX1032D96F20042R

TABLE 14B (Day 7) Evaluation of Gamma Sterilized Hoechst-L493 FiberizedMedia Platelet Yield and In Vitro Function following 7-Day Storage (NoTransfer) Platelet Count Yield Sample (×10¹¹/300 mL) (%) pH pCO₂ pO₂Control 3.88 ± 0.18 100 6.99 18 93 (+PCT-IAD) 5.0 g Hoechst/ 3.67 ± 0.0895 ± 5 6.92 22 81 L-493 7.5 g 3.48 ± 0.07 90 ± 4 6.91 20 91Hoechst/L-493 2.5 g Dowex 2.65 ± 0.22 68 ± 6 7.04 9 159 Optipore L- 493Baxter Lot FX1032 D96F20042R Shape Sample Change HSR AggregationMorphology Control 0.53 ± 0.12 0.23 ± 0.02 65 ± 5 260 5.0 g Hoechst/0.54 ± 0.00 0.30 ± 0.01 83 ± 8 266 L-493 7.5 g Hoechst/ 0.53 ± 0.09 0.29± 0.01  81 ± 13 280 L-493 2.5 g Dowex 0.23 ± 0.04 0.25 ± 0.03 33 ± 9 228Optipore L-493 Baxter Lot FX1032 D96F20042R

The results that are summarized in Tables 14A and 14B are similar to theresults that were observed in the first study. Platelets that werecontacted with the Hoechst-L493 IAD had significantly higher yields thanplatelets that were contacted with the standard removal device. Plateletyield was slightly lower for the 7.5 g Hoechst-L493 IAD relative to the5.0 g IAD. Platelets that were treated with the Hoechst-L493 IADsperformed comparably to the control platelets in all in vitro assays.Platelets that were contacted with the Hoechst-L493 IADs showed improvedperformance in the aggregation assay relative to the no-IAD control onday 7. The GMP-140 assay was not performed on day 7.

IADs that were prepared with Hoechst-L493 media (5.0, 7.0 g) resulted insuperior in vitro function when compared to standard removal devices(2.5 g loose XUS-43493) stored in contact with the platelets for 5 days.Moreover, platelets that were treated with 150 μM4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen+3.0 J/cm² UVA showedimproved in vitro function as indicated by shape change, aggregation,and GMP-140 assays when contacted with Hoechst-L493 IADs (5.0 g) for 5and 7 days. An additional study that compared 5-7 day storage forplatelets (no psoralen/UVA) with and without IAD (50.g Hoechst-L493)demonstrated that storage with an IAD may improve platelet performanceas indicated by in vitro function assays.

Example 17 Comparison of AQF Fiberized Beads vs. Free Beads in PRBCs

This study compared the removal of S-300 (N-(9-acridinyl)-β-alanine) andglutathione from PRBC using AQF fiberized vs. free beads of Ambersorb572.

PRBCs were prepared by centrifuging whole blood at 2100 rpm for 5minutes and expressing off the plasma, then adding 84 mL of Erythrosolper unit. Six ABO matched units were pooled into a 3.0 L Clintec Viaflexbag. Approximately 300 mL was transferred back to each original PL 146bag and dosed with 6.0 mL of 150 mM glutathione for a finalconcentration of 3.0 mM and 3.0 mL of 30 mM S-300 derivative for a finalconcentration of 300 μM. This was mixed manually and allowed to incubateat room temperature for 4 hours.

The PRBCs were transferred to 1 liter PL 1813 bags containing one of twoadsorption devices consisting of either 4.8 g of Ambersorb beads in AQFfiberized media (400 g/m²) or 4.8 g of loose Ambersorb beads. Thefiberized media or loose beads are enclosed in a Tekto woven mesh pouch.Duplicates were run for each adsorption device and a unit in a PL 1813bag without an adsorption device was used as a control. These werestored on a platelet shaker at room temperature for 24 hours, thentransferred to storage under static conditions at 4° C. Units weresampled prior to dosing with the S-300 derivative and glutathione, aftertreatment but prior to transfer to the adsorption devices, and at 2, 4,6, 8, 20, and 24 hours and 1, 2 and 5 weeks after transfer to theadsorption devices. These samples were prepared for HPLC analysis ofS-300 and glutathione and results are shown in table 15. Samples werealso analyzed for percentage hemolysis, ATP concentration, K⁺concentration, and pH (see table 16).

The results of this experiment suggest that the fiberized resin ispreferred over the free beads. The fiberized resin shows equivalentremoval of S-300 and glutathione with the benefit of improved red cellfunction as compared to the free beads. Table 15 shows that the removalof S-300 is similar for the two compound adsorption devices. The removalkinetics for glutathione appear to be slightly better for the fiberizedbeads but is at acceptable levels for either device after 24 hours.Table 16 shows that for red cell functional assays the fiberized resintreated samples are comparable to the control unit. Comparing the twodevices, the ATP and pH are essentially the same. The % hemolysis and K⁺levels are much higher after 24 hours for the free beads, indicatingsubstantial loss or red blood cell function.

TABLE 15 Removal of S-300 and glutathione from PRBC using Ambersorb 572beads in a fiberized media vs. free beads. Residual concentration ofcompounds in PRBC (S-300) or supernatant (glutathione) after treatment.Ambersorb 572 beads in AQF fiberized Ambersorb 572 media free beads TimeExp. 1 Exp. 2 Exp. 1 Exp. 2 Control Residual  0 33.5 33.7 31.1 32.1 32.9S-300  2 hr 4.3 6.5 5.5 6.9 42.6 μM  4 hr 2.4 2.8 3.1 2.5 54.6  6 hr 1.91.9 2.2 2.0 66.8  8 hr 2.0 2.2 2.1 1.8 74.3 20 hr 2.4 2.5 2.4 2.1 113.824 hr 2.4 2.4 2.0 2.0 120.9  1 week 13.5 13.9 14.8 15.5 150.6  2 weeks17.3 18.7 17.1 17.4 174.7  5 weeks 16.7 19 12.5 15.4 171.2 Residual  06.55 6.59 6.40 6.50 6.40 glutathione  2 hr 0.59 0.87 1.03 1.50 6.35 mM 4 hr 0.11 0.16 0.54 0.67 6.46  6 hr 0.04 0.22 0.21 0.53 6.60  8 hr 0.330.33 0.27 0.29 6.40 20 hr 0.31 0.09 0.28 0.44 6.60 24 hr 0.09 0.21 0.350.49 6.40

TABLE 16 % Hemolysis, K+, ATP, and pH comparison of PRBC treated withAmbersorb 572 as beads in a fiberized media vs. free beads. Ambersorb572 beads in AQF fiberized Ambersorb 572 media free beads Time Exp. 1Exp. 2 Exp. 1 Exp. 2 Control %  0 0.13 0.14 0.13 0.11 0.15 Hemolysis  2hr 0.14 0.16 0.61 0.81 0.14  4 hr 0.19 0.13 0.98 1.73 0.11  6 hr 0.240.17 1.49 2.73 0.12  8 hr 0.29 0.19 2.18 3.37 0.12 20 hr 0.29 0.28 6.669.56 0.14 24 hr 0.37 0.35 9.57 14.67 0.20  1 week 0.47 0.36 9.39 13.790.14  2 weeks 0.45 0.39 9.94 15.55 0.25  5 weeks 0.47 0.36 10.19 19.300.28 K⁺  0 3.51 3.52 3.52 3.49 3.57 mmol/L  4 hr 3.50 3.44 4.59 5.523.89  8 hr 3.88 3.76 6.43 7.83 4.27 20 hr 4.94 4.88 12.68 16.14 5.54 24hr 5.29 5.23 15.36 19.22 5.96  1 week 11.36 11.14 19.38 22.47 12.46  2weeks 17.70 17.76 23.92 26.52 19.35  5 weeks 32.68 31.96 34.28 35.1635.12 ATP  0 71.37 71.18 71.18 70.40 68.84 μmol/dL  1 week 63.38 62.4060.45 58.11 69.81  2 weeks 50.90 51.48 50.27 47.00 56.55  5 weeks 24.7723.79 23.01 24.18 23.40 pH  0 6.74 6.74 6.74 6.75 6.75  4 hr 6.82 6.816.81 6.80 6.73  8 hr 6.80 6.80 6.81 6.80 6.71 20 hr 6.74 6.74 6.76 6.766.64 24 hr 6.73 6.72 6.75 6.76 6.62  2 weeks 6.53 6.52 6.57 6.59 6.44  5weeks 6.40 6.39 6.46 6.48 6.26

Example 18 Effect of Mode of Agitation on the Removal of S-300 andGlutathione and on Red Blood Cell Function Using Fiberized Ambersorb 572

This study looked at the effect of the mode of agitation used during IADtreatment on removal of S-300 (N-(9-acridinyl)-β-alanine) andglutathione and on percentage hematocrit, percentage hemolysis, ATPconcentration, K⁺ concentration, and pH.

PRBCs were prepared as one pool and treated as per Example 17. Aftertreatment at room temperature for 4 hours, six units were transferred toIADs consisting of 4.8 g of Ambersorb 572 (AQF manufactured, 400 g/m²)enclosed in a Tekto woven mesh pouch. The IADs were contained in 1 literPL1813 bags. A control unit was transferred to a PL 1813 bag without anIAD. These were then treated as follows:

Agitation Type 4° C. Test during first 24 hours Storage Article at roomtemperature Condition 1 platelet shaker static (72 cycles/min) 2platelet shaker orbital shaker (72 cycles/min) (intermittent) 3 orbitalshaker static (72 cycles/min) 4 platelet rotator static (6 cycles/min) 5Nutator (3-D rotation, static 24 cycles/min) 6 static (no agitation)static 7 platelet shaker (no static (control) media or enclosure)

Units were sampled similarly to Example 17. The results indicate thatsome method of agitation is preferable for the removal of S-300 andglutathione. All modes result in equivalent removal while the orbitalshaker shows lower levels of hemolysis than the other methods. Table 17shows the S-300 and glutathione levels. Table 18 shows the red cellfunction.

TABLE 17 S-300 and glutathione levels in units treated with variousagitiation modes with AQF fiberized Ambersorb 572. Test article 1 2 3 45 6 7 RT agitation mode PS PS OS PR N static Control 4° C. conditionstatic OS static static static static static Resid-  0 34.7 33.8 34.334.2 34.7 34.8 33.9 ual  2 hr 4.4 4.7 5.0 4.0 3.6 32.5 43.9 S-300  4 hr2.9 2.5 2.2 2.7 2.1 35.9 57.4 μM  6 hr 2.3 2.1 2.3 2.1 1.8 34.7 65.1  8hr 2.5 2.0 2.0 2.0 1.7 33.6 74.4 20 hr 2.0 2.0 1.9 2.0 1.8 39.0 109.2 24hr 2.9 2.5 2.6 2.6 2.5 35.5 114.6  1 week 13.3 8.0 15.0 15.0 16.9 26.2141.0  2 weeks 16.3 8.6 17.7 17.1 18.0 22.6 160.6  5 weeks 13.7 4.0 1412.9 14.4 14.4 166.8 Resid-  0 5.97 6.02 5.98 5.96 5.92 5.92 6.93 ual  2hr 0.52 0.71 0.84 0.65 0.55 4.39 5.99 gluta-  4 hr 0.74 0.82 0.73 0.670.64 4.33 6.93 thione  6 hr 0.59 0.67 0.59 0.6 0.55 3.79 6.41 mM  8 hr0.55 0.62 0.61 0.58 0.55 3.38 6.56 20 hr 0.10 0.10 0.10 0.10 0.10 1.825.70 24 hr 0.10 0.10 0.10 0.10 0.10 1.15 5.03 PS = platelet shaker, OS =orbital shaker, PR = platelet rotator, N = Nutator

TABLE 18 % hemolysis, K⁺, ATP, and pH results with various agitationmodes using fiberized AQF Ambersorb 572. Test article 1 2 3 4 5 6 7 RTagitation mode PS PS OS PR N static Control 4° C. condition static OSstatic static static static static %  0 0.10 0.09 0.09 0.09 0.10 0.080.08 Hemo-  2 hr 0.14 0.13 0.11 0.19 0.28 0.08 0.08 lysis  4 hr 0.200.17 0.15 0.29 0.38 0.09 0.09  6 hr 0.28 0.20 0.16 0.42 0.47 0.09 0.10 8 hr 0.32 0.26 0.19 0.46 0.56 0.12 0.12 20 hr 0.63 0.43 0.27 0.97 0.990.10 0.13 24 hr 0.72 0.48 0.28 1.08 1.14 0.10 0.14  1 week 0.73 0.530.30 1.11 1.16 0.15 0.15  2 weeks 0.76 0.53 0.33 1.18 1.26 0.20 0.19  5weeks 1.18 0.75 0.40 1.18 1.34 0.34 0.24 K⁺  0 3.64 3.65 3.62 3.62 3.643.64 3.63 mmol/L  4 hr 3.84 3.76 3.80 4.01 4.15 3.67 4.02  8 hr 4.304.14 4.10 4.51 4.58 3.91 4.47 20 hr 5.46 5.14 4.93 5.89 5.78 4.57 5.4424 hr 5.92 5.59 5.20 6.30 6.14 4.83 5.85  1 week 12.88 12.80 12.38 13.1212.88 11.76 13.12  2 weeks 18.82 19.08 18.54 19.32 19.22 18.74 20.40  5weeks 31.65 31.50 31.40 31.25 30.65 30.65 24.80 pH  0 7.35 7.32 7.357.35 7.34 7.35 7.35  8 hr 7.47 7.44 7.44 7.43 7.46 7.39 7.32 24 hr 7.417.41 7.45 7.44 7.40 7.39 7.29  1 week 7.33 7.22 7.27 7.24 7.25 7.24 7.08ATP  0 75.08 76.83 77.22 75.86 75.86 77.22 76.25 μmol/  1 week 62.4062.01 65.72 63.18 62.60 66.11 66.50 dL  2 weeks 50.31 48.36 51.29 50.1249.14 53.43 51.29  5 weeks 23.40 22.43 23.99 23.79 24.18 26.13 23.99

Example 19 Removal of Activated Complement by Fiberized Ambersorb 572Treated PRBCs

This study looked at the formation of complement fragments C3a andSC5b-9 in PRBCs after treatment with an S-300 derivative and glutathionefollowed by removal using AQF fiberized media.

PRBCs were prepared as one pool and treated as per Example 17. Aftertreatment at room temperature for 4 hours, units were transferred to 1liter PL 1813 bags containing the following:

Test Article (PRBC unit #) Description 1 no IAD 2 no IAD-ice incubated 34.8 g Ambersorb IAD (400 g/m²) (no enclosure) 4 7 sheets celluloseacetate membrane (47 mm dia.)

The cellulose acetate membrane is known to cause complement activationand is used as a positive control. All but unit 2 were treated for 24hours at room temperature prior to storage under static conditions at 4°C. Unit 2 was stored at 4° C. continuously.

Three 1.5 mL samples were taken from each test article prior to IADtreatment, during treatment after 4, 8, and 24 hours, and 5 days. Eachsample was centrifuged at 2000×g for 15 minutes and 450 μL of eachsupernatant was mixed with 50 μL of cold 200 mM EDTA and vortexed. Thesewere frozen rapidly on dry ice and stored at −70° C.

Enzyme immunoassays (Quidel) were used to detect the formation ofcomplement fragments C3a and SC5b-9. Presence of these fragments are anindication of activation of the complement system. The assay involvesbinding of the target fragment by a mouse antibody which is conjugatedto Horse Radish Peroxidase (HRP) and detection using a chromogenicsubstrate of the HRP. Sample absorbance was measured against a standardcurve to calculate the fragment concentration in the sample. Sampleswere also assessed for S-300, glutathione and hemolysis similarly toExample 17.

The results indicate that complement activation is reduced in samplestreated with the Ambersorb 572 beads in AQF media relative to controls.Table 19 shows that for the controls, complement activation is lower inthe sample stored continuously at 4° C. The sample treated with the IADshowed lower levels of C3a and SC5b-9 than the control at 5 days, withC3a near the detection limit after 24 hours. Table 20 indicates thatS-300 and glutathione removal was as expected with the AQF media.

TABLE 19 Complement fragment C3a and SC5b-9 levels with varioustreatments. Test article 1 2 3 4 Treatment no IAD AQF no cellulose noIAD 4° C. pouch acetate C3a  0 411 354 391 388 ng/mL 24 hr 609 383 −1701  5 days 665 404 26 N/A SC5b-9  0 38 37 18 25 ng/mL 24 hr 120 52 2287  5 days 226 N/A 34 N/A

TABLE 20 S-300 concentration (PRBC), glutathione concentration(supernatant), and % hemolysis with various treatments. Test article 1 23 4 Treatment no IAD AQF no cellulose no IAD 4° C. pouch acetateResidual  0 30.55 30.18 29.38 29.6 S-300 24 hr 121.39 61.93 2.51 118.39μM  5 days 142.36 136.33 12.35 153.58 glutathione  5 days 7.15 7.26 0.727.07 mM %  0 0.11 0.11 0.10 0.10 Hemolysis 24 hr 0.42 0.11 0.13 0.61  5days 0.44 0.17 0.14 0.64

Example 20

Hemocompatibility enhancement of adsorbent by an inert particulatematrix. This example demonstrates that immobilization of adsorbentparticles in an inert particulate matrix enhances the hemocompatibilityof the adsorbent without substantially impacting removal of lowmolecular weight compounds. In addition, this example supports thecontention that immobilizing adsorbent particles in an inert matrix(fiber or particulate) is a general method for enhancing thehemocompatibility of the adsorbent. Results for IADs comprised ofadsorbent particles immobilized in a fiber matrix and a particulatematrix are presented below.

The media that was studied in this example is comprised of PuroliteMN-200 adsorbent particles (200-1200 μm) immobilized in ultra highmolecular weight polyethylene (UHMWPE) particulate matrix.Representative media is manufactured by Porex (Fairburn, Ga.). Disks ofimmobilized adsorbent media were formed by mixing approximately 50%(w/w) Purolite MN-200 (200-1200 μm) with 50% (w/w) UHMWPE particleshaving a similar particle size. The mixture was placed in a cylindricalcavity and heated under pressure at conditions sufficient to cause theUHMWPE particles to fuse and entrap the adsorbent particles. Theresulting disks had a diameter of 3.50 in. and were approximately 0.250in. thick. The disks weighed approximately 24 g corresponding toapproximately 12 g MN-200 in each disk. The IAD was prepared by placingthe disk of media in a plastic storage container (PL2410, BaxterHealthcare Corp.) and the entire assembly was sterilized by irradiatingwith 25-40 kGy of gamma irradiation (Sterigenics, Hayward, Calif.).

The fiber matrix IAD was comprised of Purolite MN-200 immobilized in anon-woven polyester matrix at loading of 300 g adsorbent/m².Approximately 2.5 g (adsorbent mass) of immobilized Purolite MN-200 wasplaced in a pouch constructed from 30 μm woven polyester material(Tetko, DePew, N.Y.). The pouch assembly was placed in a plastic storagecontainer (PL2410, Baxter Healthcare Corp.) and the entire assembly wassterilized by irradiating with 25-40 kGy of gamma irradiation(Sterigenics, Hayward, Calif.).

Units of ABO-matched platelet concentrates comprised of platelets(3-5×10¹¹ cells) suspended in approximately 300 mL of 35% autologousplasma, 65% platelet additive solution were obtained from SacramentoBlood Bank (Sacramento, Calif.). The platelet units were pooled anddivided before dosing each unit with 3 mL of 15 mM aminated psoralen(4′-(4-amino-2-oxa)butyl-4,5′,8-trimethyl psoralen). Each unit wassubjected to photochemical treatment by illuminating with 3.0 J/cm2 ofUVA in a UVA illumination device (Baxter Healthcare Corp.). Twophotochemically treated platelet units were transferred to the PL2410plastic storage containers with the two test SRDs. One treated unit waskept as a control and was not contacted with an IAD. All units wereplaced on a platelet shaker (Helmer, Noblesville, Ind.) at roomtemperature (22° C.) for the duration of the experiment.

The kinetics of psoralen adsorption by each of the IAD embodiments wasdetermined. Samples of each platelet unit (ca. 1 mL) were removed at 2hour intervals during the first 8 hours of storage. These samples werelater analyzed for levels of residual psoralen by High Pressure LiquidChromatography. Following 5 days of storage at room temperature, theplatelet counts, pH, and dissolved gases for each of the units wasmeasured. Platelet function was also assessed by performing in vitrotests which included: shape change, aggregation, hypotonic shockresponse, GMP-140 (p-selectin expression), and morpohology score.

The kinetics for psoralen adsorption are shown in FIG. 23. Both IADsremoved the psoralen to the limit of quantitation for the HPLC assayduring the eight hour incubation period. The particulate IAD had muchfaster adsorption kinetics due to the higher mass of adsorbent (about 12g) relative to the fiber IAD (2.5 g).

The platelet counts, pH measurements, and in vitro platelet functionassay results are summarized in Table 21. The fiber and particulatematrix IADs both gave greater than 90% recovery of platelets. Thisobservation is particularly impressive for the particulate IADconsidering that it contains about 12 g of MN-200. Note that a removaldevice that contains 2.5 g of non-immobilized adsorbent particles willtypically result in a loss of 25-35% platelets by day 5. A devicecontaining 12 g of non-immobilized particles would therefore by expectedto remove >50% of the platelets. It is obvious that immobilizing theadsorbent in the particulate matrix has drastically reduced plateletloss while the kinetics of psoralen removal are still very rapid.

Results from the in vitro platelet function studies are summarized inthe second half of Table 21. Once again, both IADs demonstratedsatisfactory performance. Hypotonic shock response was slightly higherfor the control due to a single high measurement as indicated by thelarge standard deviation. The IADs did perform better in the aggregationassay with all other assays demonstrating essentially identicalperformance.

TABLE 21 Comparison of Day 5 Platelet Yield and In Vitro PlateletFunction for Fiber Matrix and Particulate Matrix IADs Platelet CountSample (×10¹¹/300 mL) % Yield pH pCO₂ pO₂ Control 2.89 ± 0.06 100 6.9522 116 AQF Fiber Matrix IAD 2.71 ± 0.11 94 ± 4 6.96 23 112 (300 g/m²MN-200) Porex Particulate Matrix 2.61 ± 0.10 90 ± 4 6.90 22 123 IAD (50%MN-200) Sample Shape Change HSR Aggregation Morphology GMP-140 Control1.03 ± 0.33 0.71 ± 0.28 44 ± 3 308 67.0 AQF Fiber 0.96 ± 0.14 0.53 ±0.06 60 ± 0 311 63.9 Matrix IAD (300 g/m² MN-200) Porex 1.00 ± 0.05 0.42± 0.01 64 ± 1 304 66.2 Particulate Matrix IAD (50% MN-200)

The particulate matrix IAD may be further optimized from the presentconfiguration by changing the geometry of the disk to allow morecomplete penetration of the media disk with liquid. A thinner disk wouldprobably result in equivalent removal kinetics with a lower mass ofadsorbent.

In addition to optimizing the geometry of the disk, the wetting of thedevice and therefore the adsorption kinetics could be further enhancedby increasing the wetting of the media. The inherent hydrophobic natureof the UHMWPE matrix make the device wet slowly during the initial phaseof removal. Strategies that could be used to enhance wetting include theuse of wetting agents (e.g., glycerol, polyethylene oxide, polyethyleneglycol, hydrophilic polymers) or treatment with gas plasma glowdischarge. Unlike wetting agents, treatment with glow discharge can beused to directly alter the chemistry of the surface of the UHMWPE bindermatrix. Described above is a batch system. Also described herein is aflow system in accordance with U.S. Ser. No. 10/016,223 filed Dec. 10,2001, which was incorporated by reference in its entirety as if fullyput forth below including claims.

The invention claimed is:
 1. A pathogen-inactivating compound adsorptionsystem for reducing the concentration of a low molecular weightpathogen-inactivating compound in an aqueous biological compositioncontaining cellular elements, wherein the pathogen-inactivating compoundadsorption system comprises a housing compatible with the biologicalcomposition containing an adsorption medium comprising porous adsorbentparticles immobilized within a sintered matrix formed from polymericparticulate material, wherein the diameter of the adsorbent particlesranges from about 100 μm to about 1500 μm, wherein the adsorbentparticles have an affinity for said pathogen-inactivating compound,wherein the system is configured to remove said pathogen-inactivatingcompound from said biological composition in a batch process, andwherein the system is configured so that the cellular elements in thebiological composition treated with the system maintain sufficientbiological activity so that said biological composition is suitable forinfusion within a human, the system further comprising a particleretention medium downstream of the adsorption medium, wherein saidparticle retention medium retains particles shed from said adsorptionmedium.
 2. A system according to claim 1, wherein the porous adsorbentparticles have a surface area greater than about 750 m²/g, and theporous adsorbent particles are between 25 and 85 percent of the weightof the adsorption medium.
 3. A system according to claim 2, wherein theporous adsorbent particles are between 50 and 80 percent of the weightof the adsorption medium, and the adsorption medium has a particleloading of between 100 and 500 g/m².
 4. A system according to claim 3,wherein the adsorption medium has a particle loading of between 250 and350 g/m².
 5. A system according to claim 1, wherein the matrix containssaid porous adsorbent particles.
 6. A system according to claim 5,wherein the porous adsorbent particles comprise a synthetic polymericadsorbent having a porous network structure and having a surface areagreater than about 750 m²/g.
 7. A system according to claim 5, whereinthe porous adsorbent particles comprise activated carbon.
 8. A systemaccording to claim 6, wherein the porous adsorbent particles comprise apolyaromatic resin.
 9. A system according to claim 8, wherein said resinhas a pore size between about 25 and 800 Å.
 10. A system according toclaim 9, wherein said resin has a pore size between about 25 and 150 Å.11. A system according to claim 6, wherein the porous adsorbentparticles do not require prewetting before use.
 12. A system accordingto claim 6, wherein the porous adsorbent particles comprise ahypercrosslinked resin.
 13. A system according to claim 7, wherein theporous adsorbent particles comprise activated carbon having a surfacearea between about 1000 and 3000 m²/g.
 14. A system according to claim13, wherein the activated carbon is derived from a synthetic source andat least about 50% of pores of the activated carbon particles have adiameter less than about 20 Å.
 15. A system according to claim 6,wherein the diameter of the porous adsorbent particles is between about300 and 900 μm.
 16. A system according to claim 1, wherein the diameterof the porous adsorbent particles is between about 300 and 900 μm.
 17. Asystem according to claim 1, wherein the pathogen inactivating compoundcomprises a nucleic acid-binding compound.
 18. A system according toclaim 17, wherein the nucleic acid-binding compound comprises a psoralenderivative.
 19. A system according to claim 17, wherein the nucleicacid-binding compound comprises an acridine derivative.
 20. A systemaccording to claim 17, wherein the nucleic acid-binding compoundcomprises a dye.